Compositions isolated from forage grasses and methods for their use

ABSTRACT

Isolated polynucleotides encoding polypeptides active in lignin, fructan and tannin biosynthetic pathways are provided, together with expression vectors and host cells comprising such isolated polynucleotides. Methods for the use of such polynucleotides and polypeptides are also provided.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/289,757, filed Nov. 7, 2002, now U.S. Pat. No. 7,154,027, whichclaims priority to U.S. Provisional Patent Application No. 60/337,703filed Nov. 7, 2001.

TECHNICAL FIELD OF THE INVENTION

This invention relates to polynucleotides isolated from forage grasstissues, specifically from Lolium perenne (perennial ryegrass) andFestuca arundinacea (tall fescue), as well as oligonucleotide probes andprimers, genetic constructs comprising the polynucleotides, biologicalmaterials (including host cells and plants) incorporating thepolynucleotides, polypeptides encoded by the polynucleotides, andmethods for using the polynucleotides and polypeptides. Moreparticularly, the invention relates to polypeptides involved in thelignin, tannin and fructan biosynthetic pathways, and to polynucleotidesencoding such polypeptides.

BACKGROUND OF THE INVENTION

Over the past 50 years, there have been substantial improvements in thegenetic production potential of ruminant animals (sheep, cattle anddeer). Levels of meat, milk or fiber production that equal an animal'sgenetic potential may be attained within controlled feeding systems,where animals are fully fed with energy dense, conserved forages andgrains. However, the majority of temperate farming systems worldwiderely on the in situ grazing of pastures. Nutritional constraintsassociated with temperate pastures can prevent the full expression of ananimal's genetic potential. This is illustrated by a comparison betweenmilk production by North American grain-fed dairy cows and New Zealandpasture-fed cattle. North American dairy cattle produce, on average,twice the milk volume of New Zealand cattle, yet the genetic base issimilar within both systems (New Zealand Dairy Board and United StatesDepartment of Agriculture figures). Significant potential thereforeexists to improve the efficiency of conversion of pasture nutrients toanimal products through the correction of nutritional constraintsassociated with pastures.

Lignin Biosynthetic Pathway

Lignin is an insoluble polymer that serves as a matrix around thepolysaccharide components of some plant cell walls, and that isprimarily responsible for the rigidity of plant stems. Generally, thehigher the lignin content, the more rigid the plant. For example, treespecies synthesize large quantities of lignin, with lignin constituting20%-30% of the dry weight of wood. The lignin content of grasses rangesfrom 2-8% of dry weight and changes during the growing season. Inaddition to providing rigidity, lignin aids in water transport withinplants by rendering cell walls hydrophobic and water impermeable. Ligninalso plays a role in disease resistance of plants by impeding thepenetration and propagation of pathogenic agents.

Forage digestibility is affected by both lignin composition andconcentration. Lignin is largely responsible for the digestibility, orlack thereof, of forage crops, with small increases in plant lignincontent resulting in relatively high decreases (>10%) in digestibility(Buxton and Russell, Crop. Sci. 28:5358-558, 1988). For example, cropswith reduced lignin content provide more efficient forage for cattle,with the yield of milk and meat being higher relative to the amount offorage crop consumed. During normal plant growth, an increase in thematurity of the plant stem is accompanied by a corresponding increase inlignin content and composition that causes a decrease in digestibility.This change in lignin composition is to one of increasingsyringyl:guaiacyl (S:G) ratio. When deciding on the optimum time toharvest forage crops, farmers must therefore choose between a high yieldof less digestible material and a lower yield of more digestiblematerial.

Lignin is formed by polymerization of three different monolignols,para-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol that aresynthesized in a multistep pathway, with each step in the pathway beingcatalyzed by a different enzyme. The three monolignols are derived fromphenylalanine or tyrosine in a multistep process and are thenpolymerized into lignin by a free radical mechanism. Followingpolymerization, para-coumaryl alcohol, coniferyl alcohol and sinapylalcohol are converted into the p-hydroxyphenyl (H), guaiacyl (G) andsyringyl (S) units of lignin, respectively. While these three types oflignin subunits are well known, it is likely that slightly differentvariants of these subunits may be involved in the lignin biosyntheticpathway in various plants. For example, studies suggest that both freemonolignols and monolignol-4-coumarate esters may be substrates forlignin formation in grasses. The relative concentration of themonolignol residues in lignin varies among different plant species andwithin species. For example, the monolignol content for H/G/S ofgrasses, alfalfa and softwood gymnosperms is 22%/44%/34%, 7%/39%/54% and14%/80%/6%, respectively (van Soest in “Nutritional Ecology of theRuminant,” Cornell University Press, Ithaca, N.Y.). The composition oflignin may also vary among different tissues within a specific plant.

Coniferyl alcohol, para-coumaryl alcohol and sinapyl alcohol aresynthesized by similar pathways (Whetten et al., Annu. Rev. PlantPhysiol. Plant Mol. Biol. 49:585-609, 1998; Guo et al., Plant Cell13:73-88, 2001). The first step in the lignin biosynthetic pathway isthe deamination of phenylalanine or tyrosine by phenylalanineammonia-lyase (PAL) or tyrosine ammonia-lyase (TAL), respectively. Inmaize, the PAL enzyme also has TAL activity (Rosler et al., PlantPhysiol. 113:175-179, 1997). The product of TAL activity on tyrosine is4-coumarate, whereas the product of PAL activity on phenylalanine iscinnamate which is then hydroxylated by cinnamate 4-hydroxylase (C4H) toform 4-coumarate. 4-Coumarate is hydroxylated by coumarate 3-hydroxylase(C3H) to give caffeate. The newly added hydroxyl group is thenmethylated by caffeic acid O-methyl transferase (COMT) to give ferulate.Several other methylation reactions can be catalyzed by COMT, includingcaffeoylaldehyde to coniferaldehyde, and 5-hydroxyconiferaldehyde tosinapaldehyde. 4-Coumarate, caffeate and ferulate can all be conjugatedto coenzyme A by 4-coumarate: CoA ligase (4CL) to form 4-coumaryl CoA,caffeoyl CoA and feruloyl CoA, respectively. Caffeoyl CoA can then bemethylated by the enzyme caffeoyl-CoA O-methyl transferase (CAMT).

Coniferaldehyde is hydroxylated to 5-hydroxyconiferaldehyde by ferulate5-hydroxylase (F5H). Reduction of 4-coumaryl CoA, caffeoyl CoA andferuloyl-CoA to 4-coumaraldehyde, caffeoyl aldehyde and coniferaldehyde,respectively, is catalyzed by cinnamoyl-CoA reductase (CCR).Coumaraldehyde, caffeoyl aldehyde, coniferaldehyde and5-hydroxyconfieraldehyde are further reduced by the action of cinnamylalcohol dehydrogenase (CAD) to give coniferyl alcohol which is thenconverted into its glucosylated form for export from the cytoplasm tothe cell wall by coniferol glucosyl transferase (CGT). Recently asinapyl alcohol dehydrogenase (SAD) was described that convertssinapaldehyde to sinapyl alcohol (Li et al., Plant Cell 13:1567-1586,2001). Following export, the de-glucosylated form of coniferyl alcoholis obtained by the action of coniferin beta-glucosidase (CBG). Finally,polymerization of the three monolignols to provide lignin is catalyzedby phenolase (PNL), laccase (LAC) and peroxidase (PER). For a moredetailed review of the lignin biosynthetic pathway, see Whetton R andSederoff R, The Plant Cell, 7:1001-1013, 1995 and Whetten et al., Annu.Rev. Plant Physiol. Plant Mol. Biol. 49:585-609, 1998.

Both lignin levels and composition have been changed in a range of plantspecies by altering the expression of specific lignin biosyntheticenzymes. For example, anti-sense 4CL constructs in transgenic aspentrees reduced lignin content from 20 to 11% (a 45% reduction) but at thesame time increased both cellulose levels (by 15%) and growth rate (Huet al. Nature Biotechnol. 17:808-812, 1999). These trees had the samelevel of total carbon, suggesting that carbon partitioning had beenaltered. Reducing 4CL by either anti-sense or sense-suppression intobacco plants led to an accumulation of hydroxycinnamic acids in cellwalls as well as a reduction in both guaiacyl and syringyl lignin units(Kajita et al., Plant Cell. Physiol. 37:957-965, 1996). In transgenictobacco plants in which levels of C4H were reduced by anti-sense orsense suppression, total lignin content was reduced, in addition to areduction in syringyl lignin units (Sewalt et al., Plant Physiol.115:41-50, 1997). Reducing the levels of PAL in tobacco plants byanti-sense or sense-suppression reduced total lignin content but did notchange the syringyl-guaiacyl (S:G) lignin ration. In alfalfa, reducingexpression of COMT through either anti-sense or gene silencing decreasedtotal lignin by decreasing the amount of guaiacyl units and resulted ina near total loss of syringyl lignin units (Guo et al., Plant Cell13:73-88, 2001). In contrast, reducing CCOMT expression throughanti-sense or gene silencing in alfalfa plants also decreased totallignin by reducing the total amount of guaiacyl lignin units but had noeffect on the amount of syringyl lignin. Reducing CCR expression byanti-sense in tobacco plants resulted in reduced lignin content andincreased S:G ratios due to lower guaiacyl lignin units (Piquemal etal., Plant J. 13:71-83, 1998). A. thaliana plants where the F5H gene hadbeen mutated contained only traces of syringyl lignin (Marita et al.,Proc. Natl. Acad. Sci. USA 96:12323-12332, 1999).

Alteration of grass lignin composition may usefully be employed tomaintain high forage digestibility throughout the year. This is mostimportant when the plant is approaching flowering and/or duringflowering. At this time, the entire lignin biosynthetic pathway willpreferably be reduced, in particular lowering the amount of syringyllignin units, thereby lowering the S:G ratio and maintaining thedigestibility of the forage crop.

Several of the enzymes involved in the lignin biosynthetic pathway alsohave other functions within the plant. For example, PAL is a key enzymeof plant and fungi phenylpropanoid metabolism and catalyzes the firststep in phenylpropanoid metabolism. It is involved in the biosynthesisof a wide variety of secondary metabolites such as flavonoids,furanocoumar in phytoalexins and cell wall components. These compoundshave many important roles in plants during normal growth and inresponses to environmental stress. PAL catalyzes the removal of anammonia group from phenylalanine to form trans-cinnamate. PAL and therelated histidine ammonia lyase are unique enzymes which are known tohave the modified amino acid dehydroalanine (DHA) in their active site(Taylor et al., J. Biol. Chem. 265:18192-18199, 1990). Phenylalanine andhistidine ammonia-lyases (PAL) active site has a consensus ofGTITASGDLVPLSYIA. The serine residue is central to the active site, andthe region around this active site residue is well conserved (Langer etal., Biochem. 33:6462-6467, 1994).

C4H, which is a member of the cytochrome P450 monooxygenase superfamily,plays a central role in both phenylpropanoid metabolism and ligninbiosynthesis where it anchors a phenylpropanoid enzyme complex to theendoplasmic reticulum (ER). The phenylpropanoid pathway controls thesynthesis of lignin, flower pigments, signaling molecules, and a largespectrum of compounds involved in plant defense against pathogens and UVlight. This is also a branch point between general phenylpropanoidmetabolism and pathways leading to various specific end products. 4CLsare a group of enzymes necessary for maintaining a continuous metabolicflux for the biosynthesis of plant phenylpropanoids, such as lignin andflavonoids that are essential to the survival of plants, because theyserve important functions in plant growth and adaptation toenvironmental perturbations. Three isoforms of 4CL have been identifiedwith distinct substrate preference and specificities. Expression studiesin angiosperms revealed a differential behavior of the three genes invarious plant organs and upon external stimuli such as wounding and UVirradiation or upon challenge with fungi. One isoform is likely toparticipate in the biosynthetic pathway leading to flavonoids whereasthe other two are probably involved in lignin formation and in theproduction of additional phenolic compounds other than flavonoids(Ehlting et al., Plant J. 19:9-20, 1999).

F5H is involved in the phenylpropanoid biosynthesis pathway. It belongsto the CYP84 subfamily of the cytochrome P450 family and is known ascytochrome P450 84A1. F5H is one of the enzymes in the pathways leadingto the synthesis of sinapic acid esters, but also has coniferaldehydehydroxylase activity (Nair et al., Plant Physiol. 123:1623-1634, 2000).In the generalized pathway for phenylpropanoid metabolism, F5H catalyzesthe formation of 5-hydroxyferulate (a precursor of sinapate) andsinapate in turn as the precursor for sinapine and for sinapoyl CoA intwo bifurcated pathways (Chapple et al., Plant Cell 4:1413-1424, 1992).Sinapoyl CoA has been considered as the precursor for sinapyl alcohol,which is then polymerized into syringyl (S) lignin. In addition, CYP84F5H product carries out the hydroxylation of coniferaldehyde (ConAld) to5-OH ConAld (Nair et al., Plant Physiol. 123:1623-1634, 2000).

Peroxidases are heme-containing enzymes that use hydrogen peroxide asthe electron acceptor to catalyze a number of oxidative reactions. Theybelong to a superfamily consisting of 3 major classes. Class IIIconsists of the secretory plant peroxidases, which have multipletissue-specific functions in removal of hydrogen peroxide fromchloroplasts and cytosol, oxidation of toxic compounds, biosynthesis ofthe cell wall, defense responses towards wounding, indole-3-acetic acid(IAA) catabolism and ethylene biosynthesis.

Fructan Biosynthetic Pathway

Plant carbohydrates can be divided into two groups depending on theirfunction within the plant. Structural carbohydrates, such as cellulose,are usually part of the extracellular matrix. Non-structural, storagecarbohydrates act as either long- or short-term carbohydrate stores.Examples of non-structural carbohydrates include starch, sucrose andfructans.

Fructans are polymers that are stored in the vacuole and that consist oflinear and branched chains of fructose units (for review, see Vijn andSmeekens Plant Physiol. 120:351-359, 1999). They play an important rolein assimilate partitioning and possibly in stress tolerance in manyplant families. Grasses use fructans instead of starch as awater-soluble carbohydrate store (Pollock et al., in “Regulation ofprimary metabolic pathways in plants”, N. J. Kruger et al., (eds),Kluwer Academic Publishers, The Netherlands, pp 195-226, 1999).Increasing the amount of fructans and sucrose in forage crops leads toan increase in the level of water-soluble carbohydrates and therebyenhances the nutritional value of the plants. In addition, increasingthe amount of fructans in forage plants decreases methane production inanimals fed the plants, thereby leading to lower greenhouse gasemissions, and decreases urea production in animals as less protein isdegraded in the rumen (Biggs and Hancock, Trends in Plant Sci. 6:8-9,2001). Fructans have also been implicated in protecting plants againstwater deficits caused by drought or low temperatures. Introduction ofenzymes involved in the fructan biosynthetic pathway into plants that donot naturally synthesize fructans may be employed to confer coldtolerance and drought tolerance (Pilon-Smits, Plant Physiol.107:125-130, 1995).

The number of fructose units within a fructan chain is referred to asthe degree of polymerization (DP). In grasses, fructans of DP 6-10 arecommon. Such fructans of low DP are naturally sweet and are therefore ofinterest for use as sweeteners in foodstuffs. Long fructan chains formemulsions with a fat-like texture and a neutral taste. The humandigestive system is unable to degrade fructans, and fructans of high DPmay therefore be used as low-calorie food ingredients. Over-expressionof enzymes involved in the fructan biosynthetic pathway may be usefullyemployed to produce quantities of fructans that can be purified forhuman consumption.

Five major classes of structurally different fructans have beenidentified in plants, with each class showing a different linkage of thefructosyl residues. Fructans found in grasses are of the mixed levanclass, consisting of both (2-1)- and (2-6)-linked beta-D-fructosyl units(Pollock et al., in “Regulation of primary metabolic pathways inplants”, N. J. Kruger et al., (eds), Kluwer Academic Publishers, TheNetherlands, pp 195-226, 1999). Fructans are synthesized by the actionof fructosyltransferase enzymes on sucrose in the vacuole. These enzymesare closely related to invertases, enzymes that normally hydrolyzesucrose.

Grasses use two fructosyltransferase enzymes to synthesize fructans,namely sucrose:sucrose 1-fructosyltransferase (1-SST) andsucrose:fructan 6-fructosyltransferase (6-SFT) (Pollock et al., in“Regulation of primary metabolic pathways in plants”, N. J. Kruger etal., (eds), Kluwer Academic Publishers, The Netherlands, pp 195-226,1999). 1-SST is a key enzyme in plant fructan biosynthesis, while 6-SFTcatalyzes the formation and extension of beta-2,6-linked fructans thatis typically found in grasses. Specifically, 1-SST catalyzes theformation of 1-kestose plus glucose from sucrose, while 6-SFT catalyzesthe formation of bifurcose plus glucose from sucrose plus 1-kestose andalso the formation of 6-kestose plus glucose from sucrose. Both enzymescan modify 1-kestose, 6-kestose and bifurcose further by addingadditional fructose molecules. Over-expression of both 1-SST and 6-SFTin the same plant may be employed to produce fructans for use in humanfoodstuffs (Sevenier et al., Nature Biotechnol. 16:843-846; Hellwege etal., Proc. Nat. Acad. Sci. USA 97:8699-8704, 2000).

The synthesis of sucrose from photosynthetic assimilates in plants, andtherefore the availability of sucrose for use in fructan formation, iscontrolled, in part, by the enzymes sucrose phosphate synthase (SPS) andsucrose phosphate phosphatase (SPP). Sucrose plays an important role inplant growth and development, and is a major end product ofphotosynthesis. It also functions as a primary transport sugar and insome cases as a direct or indirect regulator of gene expression (for areview see Smeekens, Curr. Opin. Plant Biol. 1:230-234, 1998). SPSregulates the synthesis of sucrose by regulating carbon partitioning inthe leaves of plants and therefore plays a major role as a limitingfactor in the export of photoassimilates out of the leaf. The activityof SPS is regulated by phosphorylation and moderated by concentration ofmetabolites and light (Huber et al., Plant Physiol. 95:291-297, 1991).Specifically, SPS catalyzes the transfer of glucose from UDP-glucose tofructose-6-phosphate, thereby forming sucrose-6-phosphate (Suc-6-P).Suc-6-P is then dephosphorylated by SPP to form sucrose (Lunn et al.,Proc. Natl. Acad. Sci. USA 97:12914-12919, 2000). The enzymes SPS andSPP exist as a heterotetramer in the cytoplasm of mesophyll cells inleaves, with SPP functioning to regulate SPS activity. SPS is alsoimportant in ripening fruits, sprouting tubers and germinating seeds(Laporte et al., Planta 212:817-822, 2001).

Once in the vacuole, sucrose can be converted into fructan byfructosyltransferases as discussed above, or hydrolyzed into glucose andfructose by the hydrolase enzymes known as invertases (Sturm, PlantPhysiol. 121:1-7, 1999). There are several different types ofinvertases, namely extracellular (cell wall), vacuolar (soluble acid)and cytoplasmic, with at least two isoforms of each type of invertasenormally being found within a plant species. In addition to havingdifferent subcellular locations, the different types of invertases havedifferent biochemical properties. For example, soluble and cell wallinvertases operate at acidic pH, whereas cytoplasmic invertases work ata more neutral or alkaline pH. Invertases are believed to regulate theentry of sucrose into different utilization pathways (Grof and Campbell,Aust. J. Plant Physiol. 28:1-12, 2001). Reduced vacuolar or cytoplasmicinvertase activity in sink tissues may increase the level ofwater-soluble carbohydrates in plants. Plants contain several isoformsof cell wall invertases (CWINV), which accumulate as insoluble proteins.CWINV plays an important role in phloem unloading and in stressresponse. It hydrolyzes terminal non-reducing beta-D-fructofuranosideresidues in beta-D-fructo-furanosides.

Another enzyme that acts upon sucrose in plants is soluble sucrosesynthase (SUS). Recent results indicate that SUS is localized in thecytosol, associated with the plasma membrane and the actin cytoskeleton.Phosphorylation of SUS is one of the factors controlling localization ofthe enzyme (Winter and Huber, Crit. Rev. Biochem. Mol. Biol. 35:253-89,2000). It catalyzes the transfer of glucose from sucrose to UDP,yielding UDP-glucose and fructose. Increasing the amount of SUS in aplant increases the amount of cellulose synthesis, whereas decreasingSUS activity should increase fructan levels. Increased SUS concentrationmay also increase the yield of fruiting bodies. SUS activity is highestin carbon sink tissues in plants and low in photosynthetic sourcetissues, and studies have indicated that SUS is the mainsucrose-cleaving activity in sink tissues. Grasses have two isoforms ofSUS that are encoded by separate genes. These genes are differentiallyexpressed in different tissues.

Tannin Biosynthetic Pathway

Condensed tannins are polymerized flavonoids. More specifically, tanninsare composed of catechin 4-ol and catechin monomer units, and are storedin the vacuole. In many temperate forage crops, such as ryegrass andfescue, foliar tissues are tannin-negative. This leads to a high initialrate of fermentation when these crops are consumed by ruminantlivestock, resulting in both protein degradation and production ofammonia by the livestock. These effects can be reduced by the presenceof low to moderate levels of tannin. In certain other plant species, thepresence of high levels of tannins reduces palatability and nutritivevalue. Introduction of genes encoding enzymes involved in thebiosynthesis of condensed tannins into a plant may be employed tosynthesize flavonoid compounds that are not normally made in the plant.These compounds may then be isolated and used for treating human oranimal disorders or as food additives.

Much of the biosynthetic pathway for condensed tannins is shared withthat for anthocyanins, which are pigments responsible for flower color.Therefore, modulation of the levels of enzymes involved in the tanninbiosynthetic pathway may be employed to alter the color of foliage andthe pigments produced in flowers.

Most tannins described to date contain pro-cyanidin units derived fromdihydroquercetin and pro-delphinidin units derived fromdihydromyricetin. However, some tannins contain pro-pelargonidin unitsderived from dihydrokaempferol. The initial step in the tanninbiosynthetic pathway is the condensation of coumaryl CoA with malonylCoA to give naringenin-chalcone, which is catalyzed by the enzymechalcone synthase (CHS). The enzyme chalcone isomerase (CHI) catalyzesthe isomerization of naringenin chalcone to naringenin (also known asflavanone), which is then hydroxylated by the action of the enzymeflavonone 3-beta-hydroxylase (F3βH) to give dihydrokaempferol. Theenzyme flavonoid 3′-hydroxylase (F3′OH) catalyzes the conversion ofdihydrokaempferol to dihydroquercetin, which in turn can be convertedinto dihydromyricetin by the action of flavonoid 3′5′-hydroxylase(F3′5′OH). The enzyme dihydroflavonol-4-reductase (DFR) catalyzes thelast step before dihydrokaempferol, dihydroquercetin anddihydromyricetin are committed for either anthocyanin (flower pigment)or proanthocyanidin (condensed tannin) formation. DFR also convertsdihydrokaempferol to afzelchin-4-ol, dihydroquercetin to catechin-4-ol,and dihydromyricetin to gallocatechin-4-ol, probably by the action ofmore than one isoform. For a review of the tannin biosynthetic pathway,see, Robbins M. P. and Morris P. in Metabolic Engineering of PlantSecondary Metabolism, Verpoorte and Alfermann (eds), Kluwer AcademicPublishers, the Netherlands, 2000.

While polynucleotides encoding some of the enzymes involved in thelignin, fructan and tannin biosynthetic pathways have been isolated forcertain species of plants, genes encoding many of the enzymes in a widerange of plant species have not yet been identified. Thus there remainsa need in the art for materials useful in the modification of lignin,fructan and tannin content and composition in plants, and for methodsfor their use.

SUMMARY OF THE INVENTION

The present invention provides enzymes involved in the lignin, fructanor tannin biosynthetic pathways that are encoded by polynucleotidesisolated from forage grass tissues. The polynucleotides were isolatedfrom Lolium perenne (perennial ryegrass) and Festuca arundinacea (tallfescue) tissues taken at different times of the year, specifically inwinter and spring, and from different parts of the plants, including:leaf blades, leaf base, pseudostems, floral stems, roots, inflorescencesand stems. The present invention also provides genetic constructs,expression vectors and host cells comprising the inventivepolynucleotides, and methods for using the inventive polynucleotides andgenetic constructs to modulate the biosynthesis of lignins, fructans andtannins.

In specific embodiments, the isolated polynucleotides of the presentinvention comprise a sequence selected from the group consisting of: (a)SEQ ID NO: 1-62 and 125-162; (b) complements of SEQ ID NO: 1-62 and125-162; (c) reverse complements of SEQ ID NO: 1-62 and 125-162; (d)reverse sequences of SEQ ID NO: 1-62 and 125-162; (e) sequences having a99% probability of being functionally or evolutionarily related to asequence of (a)-(d), determined as described below; and (f) sequenceshaving at least 75%, 80%, 90% or 98% identity to a sequence of (a)-(d),the percentage identity being determined as described below.Polynucleotides comprising at least a specified number of contiguousresidues (“x-mers”) of any of SEQ ID NO: 1-62 and 125-162; andoligonucleotide probes and primers corresponding to SEQ ID NO: 1-62 and125-162 are also provided. All of the above polynucleotides are referredto herein as “polynucleotides of the present invention.”

In further aspects, the present invention provides isolated polypeptidescomprising an amino acid sequence of SEQ ID NO: 63-124 and 163-190,together with polypeptides comprising a sequence having at least 75%,80%, 90% or 98% identity to a sequence of SEQ ID NO: 63-124 and 163-190,wherein the polypeptide possesses the same functional activity as thepolypeptide comprising a sequence of SEQ ID NO: 63-124 and 163-190. Thepresent invention also contemplates isolated polypeptides comprising atleast a functional portion of a polypeptide comprising an amino acidsequence selected from the group consisting of: (a) SEQ ID NO: 63-124and 163-190; and (b) sequences having at least 75%, 80%, 90% or 98%identity to a sequence of SEQ ID NO: 63-124 and 163-190.

In another aspect, the present invention provides genetic constructscomprising a polynucleotide of the present invention, either alone, incombination with one or more of the inventive sequences, or incombination with one or more known polynucleotides.

In certain embodiments, the present invention provides geneticconstructs comprising, in the 5′-3′ direction: a gene promoter sequence;an open reading frame coding for at least a functional portion of apolypeptide of the present invention; and a gene termination sequence.An open reading frame may be orientated in either a sense or anti-sensedirection. Genetic constructs comprising a non-coding region of apolynucleotide of the present invention or a polynucleotide sequencecomplementary to a non-coding region, together with a gene promotersequence and a gene termination sequence, are also provided. Preferably,the gene promoter and termination sequences are functional in a hostcell, such as a plant cell. Most preferably, the gene promoter andtermination sequences are those of the original enzyme genes but othersgenerally used in the art, such as the Cauliflower Mosaic Virus (CMV)promoter, with or without enhancers, such as the Kozak sequence or Omegaenhancer, and Agrobacterium tumefaciens nopalin synthase terminator maybe usefully employed in the present invention. Tissue-specific promotersmay be employed in order to target expression to one or more desiredtissues. The construct may further include a marker for theidentification of transformed cells.

In a further aspect, transgenic cells, such as transgenic plant cells,comprising the constructs of the present invention are provided,together with tissues and plants comprising such transgenic cells, andfruits, seeds and other products, derivatives, or progeny of suchplants.

In yet another aspect, methods for modulating the lignin, fructan ortannin content and composition of a target organism, such as a plant,are provided, such methods including stably incorporating into thegenome of the target plant a genetic construct comprising apolynucleotide of the present invention. In a preferred embodiment, thetarget plant is a forage grass, preferably selected from the groupconsisting of Lolium and Festuca species, and most preferably from thegroup consisting of Lolium perenne and Festuca arundinacea. In a relatedaspect, a method for producing a plant having altered lignin, fructan ortannin composition is provided, the method comprising transforming aplant cell with a genetic construct comprising of the present inventionto provide a transgenic cell, and cultivating the transgenic cell underconditions conducive to regeneration and mature plant growth.

In yet a further aspect, the present invention provides methods formodifying the activity of an enzyme in a target organism, such as aplant, comprising stably incorporating into the genome of the targetorganism a genetic construct of the present invention. In a preferredembodiment, the target plant is a forage grass, preferably selected fromthe group consisting of Lolium and Festuca species, and most preferablyfrom the group consisting of Lolium perenne and Festuca arundinacea.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the activity of recombinant LpSPP (SEQ ID NO: 8) and FaSPP(SEQ ID NO 7) on dephosphorylating Suc-6-P and Fru-6-P. The pET41aextract was the vector control.

FIG. 2 shows the peroxidase activity of FaPER3 (SEQ ID NO: 50) andLpPER5 (SEQ ID NO: 52) as determined by oxidation of ABTS. Horseradishperoxidase of known activity (Sigma, St Louis, Mo.) was used as apositive control and boiled samples as a negative control.

FIG. 3 shows PCR verification of transgenic N. benthamiana plantstransformed with Lp6-SFT1 (SEQ ID NO: 3). Genomic DNA was isolated fromkanamycin resistant T2 N. benthamiana plants and the Lp6-SFT1 fragmentwas amplified using specific PCR primers.

FIG. 4 shows PCR verification of transgenic N. benthamiana plantstransformed with Lp1-SST (SEQ ID NO: 1). Genomic DNA was isolated fromkanamycin resistant T2 N. benthamiana plants and the Lp1-SST fragmentwas amplified using specific PCR primers. Plant number 5 is anon-transgenic control.

FIG. 5 shows the fructan level in transgenic N. benthamiana linestransformed with Lp6-SFT1 (SEQ ID NO: 3) and Lp1-SST (SEQ ID NO: 1).

FIG. 6 shows the sucrose synthesizing activity of FaSPS-N (SEQ ID NO: 9)with and without SPP (SEQ ID NO: 8) in mammalian cell extracts. Thenon-transfected cells are controls.

FIG. 7 shows the sucrose cleaving activity of FaSUS1 (SEQ ID NO: 13) inmammalian cell extracts.

FIG. 8 shows the invertase activity for vacuolar invertase (LpSINV1, SEQID NO: 25) and two cell wall invertases (LpCWINV1 and FaCWINV4, SEQ IDNO: 17 and 19); absence of invertase activity from an empty vector(pPICZαA) control is also shown.

DETAILED DESCRIPTION OF THE INVENTION

The polypeptides of the present invention, and the polynucleotidesencoding the polypeptides, have activity in lignin, fructan and tanninbiosynthetic pathways in plants. Using the methods and materials of thepresent invention, the lignin, fructan and/or tannin content of a plantmay be modulated by modulating expression of polynucleotides of thepresent invention, or by modifying the polynucleotides or polypeptidesencoded by polynucleotides. The isolated polynucleotides andpolypeptides of the present invention may thus be usefully employed inthe correction of nutritional imbalances associated with temperatepastures and to increase the yield of animal products from pastures.

The lignin, fructan and/or tannin content of a target organism, such asa plant, may be modified, for example, by incorporating additionalcopies of genes encoding enzymes involved in the lignin, fructan ortannin biosynthetic pathways into the genome of the target plant.Similarly, a modified lignin, fructan and/or tannin content can beobtained by transforming the target plant with anti-sense copies of suchgenes. In addition, the number of copies of genes encoding for differentenzymes in the lignin, fructan and tannin biosynthetic pathways can bemanipulated to modify the relative amount of each monomer unitsynthesized, thereby leading to the formation of lignins, fructans ortannins having altered composition.

The present invention thus provides methods for modulating thepolynucleotide and/or polypeptide content and composition of anorganism, such methods involving stably incorporating into the genome ofthe organism a genetic construct comprising one or more polynucleotidesof the present invention. In one embodiment, the target organism is aplant species, preferably a forage plant, more preferably a grass of theLolium or Festuca species, and most preferably Lolium perenne or Festucaarundinacea. In related aspects, methods for producing a plant having analtered genotype or phenotype is provided, such methods comprisingtransforming a plant cell with a genetic construct of the presentinvention to provide a transgenic cell, and cultivating the transgeniccell under conditions conducive to regeneration and mature plant growth.Plants having an altered genotype or phenotype as a consequence ofmodulation of the level or content of a polynucleotide or polypeptide ofthe present invention compared to a wild-type organism, as well ascomponents (seeds, etc.) of such plants, and the progeny of such plants,are contemplated by and encompassed within the present invention.

The isolated polynucleotides of the present invention have utility ingenome mapping, in physical mapping, and in positional cloning of genes.Additionally, the polynucleotide sequences identified as SEQ ID NO: 1-62and 125-162 and their variants, may be used to design oligonucleotideprobes and primers. Oligonucleotide probes and primers have sequencesthat are substantially complementary to the polynucleotide of interestover a certain portion of the polynucleotide. Oligonucleotide probesdesigned using the polynucleotides of the present invention may beemployed to detect the presence and examine the expression patterns ofgenes in any organism having sufficiently similar DNA and RNA sequencesin their cells using techniques that are well known in the art, such asslot blot DNA hybridization techniques. Oligonucleotide primers designedusing the polynucleotides of the present invention may be used for PCRamplifications. Oligonucleotide probes and primers designed using thepolynucleotides of the present invention may also be used in connectionwith various microarray technologies, including the microarraytechnology of Affymetrix (Santa Clara, Calif.).

In a first aspect, the present invention provides isolatedpolynucleotide sequences identified in the attached Sequence Listing asSEQ ID NO: 1-62 and 125-162, and polypeptide sequences identified in theattached Sequence Listing as SEQ ID NO: 63-124 and 163-190. Thepolynucleotides and polypeptides of the present invention havedemonstrated similarity to the following polypeptides that are known tobe involved in lignin, fructan and tannin biosynthetic processes:

TABLE 1 SEQ ID NO SEQ ID NO Polynucleotide Polypeptide CategoryDescription  1 and 125  63 and 163 Fructan Homolog of Sucrose:Sucrose1-fructosyl- biosynthesis transferase (1-SST) isolated from Festucaarundinacea. They contain a typical signature of the glycosyl hydrolasesfamily 32 (amino acid residues 120 to 133). The glycosyl hydrolasesfamily 32 domain signature has a consensus of HYQPxxH/NxxNDPNG, where Dis the active site residue (Henrissat, Biochem. J. 280: 309-316, 1991). 2 64 Fructan Homolog of Sucrose:Sucrose 1-fructosyl- biosynthesistransferase (1-SST) isolated from Festuca arundinacea. It contains atypical signature of the glycosyl hydrolases family 32 (amino acidresidues 120 to 133). The glycosyl hydrolases family 32 domain signaturehas a consensus of HYQPxxH/NxxNDPNG, where D is the active site residue(Henrissat, Biochem. J. 280: 309-316, 1991).  3 and 126  65 and 164Fructan Homolog of Sucrose:fructan 6-fructosyl- biosynthesis transferase(6-SFT) isolated from Festuca arundinacea. They contain a typicalsignature of the glycosyl hydrolases family 32 (amino acid residues 90to 564). The glycosyl hydrolases family 32 domain signature has aconsensus of HYQPxxH/NxxNDPNG, where D is the active site residue(Henrissat, Biochem. J. 280: 309-316, 1991).  4 and 127  66 and 165Fructan Homolog of Sucrose:fructan 6-fructosyl- biosynthesis transferase(6-SFT) isolated from Lolium perenne. They contain a typical signatureof the glycosyl hydrolases family 32 (amino acid residues 96 to 107).The glycosyl hydrolases family 32 domain signature has a consensus ofHYQPxxH/NxxNDPNG, where D is the active site residue (Henrissat,Biochem. J. 280: 309-316, 1991).  5 67 Fructan Homolog ofsucrose:fructan 6-fructosyl- biosynthesis transferase (6-SFT) isolatedfrom Festuca arundinacea.  6 and 128  68 and 166 Fructan Homolog ofSucrose:fructan 6-fructosyl- biosynthesis transferase (6-SFT) isolatedfrom Lolium perenne. They contain a typical signature of the glycosylhydrolases family 32 (amino acid residues 90 to 103). The glycosylhydrolases family 32 domain signature has a consensus ofHYQPxxH/NxxNDPNG, where D is the active site residue (Henrissat,Biochem. J. 280: 309-316, 1991).  7 and 129 69 Fructan Homolog ofSucrose-6-phosphate phospho- biosynthesis hydrolase (SPP; EC 3.1.3.24)isolated from Festuca arundinacea. This enzyme belongs to thesuperfamily of hydrolases, and has the three conserved motifs found inthese proteins (Galperin and Koonin, Trends Biochem Sci. 23: 127-129,1998). Motif I (amino acid residues 10 to 19) contains conserved Asp anda Thr residues, motif II (amino acid residues 48 to 53) contains aconserved Thr residue, and Motif III (residues 167 to 220) containsconserved Lys (position 167) and Asp residues (position 202 and 206).These conserved amino acid residues are required for activity of theenzyme.  8 70 Fructan Homolog of Sucrose-6-phosphate phospho-biosynthesis hydrolase (SPP; EC 3.1.3.24) isolated from Lolium perenne.This enzyme belongs to the superfamily of hydrolases, and has the threeconserved motifs found in these proteins (Galperin and Koonin, TrendsBiochem Sci. 23: 127-129, 1998). Motif I (residues 10 to 19) containsconserved Asp and Thr residues, motif II (amino acid residues 48 to 53)contains a conserved Thr residue, and Motif III (amino acid residues 167to 220) contains conserved Lys (position 167) and Asp residues (position202 and 206). These conserved amino acid residues are required foractivity of the enzyme.  9 and 130 71 Fructan Homolog of sucrosephosphate synthase (SPS- biosynthesis 1) isolated from Festucaarundinacea. 10 and 131  72 and 167 Fructan Homolog of sucrose phosphatesynthase (SPS- biosynthesis 1) isolated from Lolium perenne and that isinvolved in the sucrose synthesis pathway. 11 and 132  73 and 168Fructan Homolog of sucrose phosphate synthase (SPS- biosynthesis N)isolated from Lolium perenne and that is involved in the sucrosesynthesis pathway. 12 and 133  74 and 169 Fructan Homolog of sucrosesynthase (SuS) isolated biosynthesis from Lolium perenne. Thesemolecules contain a leucine zipper motif in amino acid position 191 to213. Leucine zipper motifs are present in many gene regulatory proteins(Landschulz et at., Science 240: 1759-1764, 1988). 13 75 Fructan Homologof sucrose synthase (SuS) isolated biosynthesis from Festucaarundinacea. This molecule contains a leucine zipper motif in amino acidposition 191 to 213. Leucine zipper motifs are present in many generegulatory proteins (Landschulz et al., Science 240: 1759-1764, 1988).14 and 134  76 and 170 Fructan Homolog of sucrose synthase (SuS)isolated biosynthesis from Lolium perenne. 15 77 Fructan Homolog ofsucrose synthase (SuS) isolated biosynthesis from Festuca arundinacea.16 and 135  78 and 171 Fructan Homologue of cell wall invertase (CWINV)biosynthesis isolated from Festuca arundinacea that belongs to thefamily 32 of glycosyl hydrolases. These molecules contain a conservedpeptide domain in amino acid residues 139 to 144 and 242-247,respectively. The consensus peptide domain of invertases is(WVYL)EC(PIL)D (LFI) with the conserved Cys residue part of thecatalytic domain (Sturm, Plant Physiol. 121: 1-7, 1999). 17 79 FructanHomolog of cell wall invertase (CWINV) biosynthesis isolated from Loliumperenne that belongs to the family 32 of glycosyl hydrolases. Thismolecule contains a conserved pentapeptide bF-motif at amino acidresidues 70 to 74 and a peptide domain in amino acid residues 250 to255. The consensus peptide domain of invertases is (WVYL)EC(PIL)D(LFI)with the conserved Cys residue part of the catalytic domain (Sturm,Plant Physiol. 121: 1-7, 1999). It also contains a glycosyl hydrolasesfamily 32 signature region at amino acids 61 to 74 that contains aconserved His residue important in the catalytic reaction (Reddy andMaley, J. Biol. Chem. 265: 10817-10120, 1990). 18 and 136  80 and 172Fructan Homolog of cell wall invertase (CWINV) biosynthesis isolatedfrom Lolium perenne that belongs to the family 32 of glycosylhydrolases. 19 81 Fructan Homolog of cell wall invertase (CWINV)biosynthesis isolated from Festuca arundinacea that belongs to thefamily 32 of glycosyl hydrolases. This molecule contains a conservedpentapeptide bF-motif at amino acid residues 60 to 64. The consensuspeptide domain of invertases is (WVYL)EC(PIL)D(LFI) with the conservedCys residue part of the catalytic domain (Sturm, Plant Physiol. 121:1-7, 1999). It also contains a glycosyl hydrolases family 32 signatureregion at amino acids 51 to 64 that contains a conserved His residueimportant in the catalytic reaction (Reddy and Maley, J. Biol. Chem.265: 10817-10120, 1990). A signal peptide is present in amino acidresidues 1 to 24. 20 and 137  82 and 173 Fructan Homolog of cell wallinvertase (CWINV) biosynthesis isolated from Festuca arundinacea thatbelongs to the family 32 of glycosyl hydrolases. These molecules containa peptide domain in amino acid residues 61 to 66and 242-247,respectively. The consensus peptide domain of invertases is(WVYL)EC(PIL)D(LFI) with the conserved Cys residue part of the catalyticdomain (Sturm, Plant Physiol. 121: 1-7, 1999). 21 83 Fructan Homolog ofcell wall invertase (CWINV) biosynthesis isolated from Festucaarundinacea that belongs to the family 32 of glycosyl hydrolases. Thismolecule contains a conserved pentapeptide bF-motif at amino acidresidues 73 to 77 and a peptide domain in amino acid residues 253 to258. The consensus peptide domain of invertases is (WVYL)EC(PIL)D-(LFI)with the conserved Cys residue part of the catalytic domain (Sturm,Plant Physiol. 121: 1-7, 1999). It also contains a glycosyl hydrolasesfamily 32 signature region at amino acid 64 to 77 that contains aconserved His residue important in the catalytic reaction (Reddy andMaley, J. Biol. Chem. 265: 10817-10120, 1990). 22 and 138  84 and 174Fructan Homolog of cell wall invertase (CWINV) biosynthesis isolatedfrom Lolium perenne that belongs to the family 32 of glycosylhydrolases. These molecules contain a peptide domain in amino acidresidues 174 to 179 and 234 to 239, respectively. The consensus peptidedomain of invertases is (WVYL)EC-(PIL)D(LFI) with the conserved Cysresidue part of the catalytic domain (Sturm, Plant Physiol. 121: 1-7,1999). 23 85 Fructan Homolog of cell wall invertase (CWINV) biosynthesisisolated from Festuca arundinacea that belongs to the family 32 ofglycosyl hydrolases. This molecule contains a conserved pentapeptidebF-motif at amino acid residues 56 to 60. The consensus peptide domainof invertases is (WVYL)EC(PIL)D(LFI) with the conserved Cys residue partof the catalytic domain (Sturm, Plant Physiol. 121: 1-7, 1999). It alsocontains a glycosyl hydrolases family 32 signature region at amino acid47 to 60 that contains a conserved His residue that is important in thecatalytic reaction (Reddy and Maley, J. Biol. Chem. 265: 10817-10120,1990). A signal peptide is present in amino acid residues 1 to 22. 24and 139  86 and 175 Fructan Homolog of cell wall invertase (CWINV)biosynthesis isolated from Lolium perenne that belongs to the family 32of glycosyl hydrolases. These molecules contain a conserved pentapeptidebF-motif at amino acid residues 244 to 249. The consensus peptide domainof invertases is (WVYL)EC(PIL)D(LFI) with the conserved Cys residue partof the catalytic domain (Sturm, Plant Physiol. 121: 1-7, 1999). Theyalso contain a glycosyl hydrolases family 32 signature region at aminoacid 56 to 69 that contains a conserved His residue that is important inthe catalytic reaction (Reddy and Maley, J. Biol. Chem. 265:10817-10120, 1990). A signal peptide is present in amino acid residues 1to 25. 25 and 140  87 and 176 Fructan Homolog of vacuolar invertase(SINV) isolated biosynthesis from Lolium perenne that belongs to thefamily 32 of glycosyl hydrolases. These molecules contain a conservedpentapeptide bF-motif at amino acid residues 136 to 140 and 138 to 142,respectively and a peptide domain in amino acid residues 317 to 322 and319 to 324, respectively. The consensus peptide domain of invertases is(WVYL)EC(PIL)D(LFI) with the conserved Cys residue part of the catalyticdomain (Sturm, Plant Physiol. 121: 1-7, 1999). It also contains aglycosyl hydrolases family 32 signature region at amino acid 127 to 140and 129 to 142 that contains a conserved His residue that is importantin the catalytic reaction (Reddy and Maley, J. Biol. Chem. 265:10817-10120, 1990). 26 and 141  88 and 177 Fructan Homolog of invertase(SINV) isolated from biosynthesis Lolium perenne that belongs to thefamily 32 of glycosyl hydrolases. These molecules contain a peptidedomain in amino acid residues 143 to 148 and 184 to 189, respectively.The consensus peptide domain of invertases is (WVYL)EC(PIL)D(LFI) withthe conserved Cys residue part of the catalytic domain (Sturm, PlantPhysiol. 121: 1-7, 1999). 27 89 Lignin/Tannin Homolog of 4-Coumarate:CoAligase (4CL, biosynthesis EC 6.2.1.12) isolated from Lolium perenne Themolecule has two conserved AMP binding regions at amino acid residues182 to 193 and 383 to 389 (Hu et al., Proc. Natl. Acad. Sci. USA 95:5407-5412, 1998). The AMP-binding domain signature consists of twobinding site motifs. The consensus of the first motif is LPYSSGTTGLPK(Etchegaray et al., Biochem. Mol. Biol. Int. 44: 235-243, 1998). Theregion very rich in glycine, serine, and threonine followed by aconserved lysine. In most of these proteins, the residue that followsthe Lys at the end of the pattern is a Gly. The second motif consensussequence is GEIC(V/I)RG (Hu et al., Proc. Natl. Acad. Sci. USA 95:5407-5412, 1998). 28 and 142 90 Lignin/Tannin Homolog of 4-Coumarate:CoAligase (4CL, biosynthesis EC 6.2.1.12) isolated from Lolium perenne. Themolecule has two conserved AMP binding regions at amino acid residues195 to 206 and 395 to 401 (Hu et al., Proc. Natl. Acad. Sci. USA 95:5407-5412, 1998). The AMP-binding domain signature consists of twobinding site motifs. The consensus of the first motif is LPYSSGTTGLPK(Etchegaray et al., Biochem. Mol. Biol. Int. 44: 235-243, 1998). Theregion very rich in glycine, serine, and threonine followed by aconserved lysine. In most of these proteins, the residue that followsthe Lys at the end of the pattern is a Gly. The second motif consensussequence is GEIC(V/I)RG (Hu et al., Proc. Natl. Acad. Sci. USA 95:5407-5412, 1998). 29 91 Lignin/Tannin Homolog of 4-Coumarate:CoA ligase(4CL, biosynthesis EC 6.2.1.12) isolated from Festuca arundinacea. Themolecule has two conserved AMP binding regions at amino acid residues195 to 206 and 395 to 401 (Hu et al., Proc. Natl. Acad. Sci. USA 95:5407-5412, 1998). The AMP-binding domain signature consists of twobinding site motifs. The consensus of the first motif is LPYSSGTTGLPK(Etchegaray et al., Biochem. Mol. Biol. Int. 44: 235-243, 1998). Theregion very rich in glycine, serine, and threonine followed by aconserved lysine. In most of these proteins, the residue that followsthe Lys at the end of the pattern is a Gly. The second motif consensussequence is GEIC(V/I)RG (Hu et al., Proc. Natl. Acad. Sci. USA 95:5407-5412, 1998). 30 and 143  92 and 178 Lignin/Tannin Homolog of4-Coumarate:CoA ligase (4CL, biosynthesis EC 6.2.1.12) isolated fromLolium. The molecules have two conserved AMP binding regions at aminoacid residues 194 to 205 and 394 to 400 (Hu et al., Proc. Natl. Acad.Sci. USA 95: 5407-5412, 1998). The AMP-binding domain signature consistsof two binding site motifs. The consensus of the first motif isLPYSSGTTGLPK (Etchegaray et al., Biochem. Mol. Biol. Int. 44: 235-243,1998). The region very rich in glycine, serine, and threonine followedby a conserved lysine. In most of these proteins, the residue thatfollows the Lys at the end of the pattern is a Gly. The second motifconsensus sequence is GEIC(V/I)RG (Hu et al., Proc. Natl. Acad. Sci. USA95: 5407-5412, 1998). 31 93 Lignin/Tannin Homolog of 4-Coumarate:CoAligase (4CL, biosynthesis EC 6.2.1.12) isolated from Festucaarundinacea. The molecule has two conserved AMP binding regions at aminoacid residues 194 to 206 and 482 to 490 (Hu et al., Proc. Natl. Acad.Sci. USA 95: 5407-5412, 1998). The AMP-binding domain signature consistsof two binding site motifs. The consensus of the first motif isLPYSSGTTGLPK (Etchegaray et al., Biochem. Mol. Biol. Int. 44: 235-243,1998). The region very rich in glycine, serine, and threonine followedby a conserved lysine. In most of these proteins, the residue thatfollows the Lys at the end of the pattern is a Gly. The second motifconsensus sequence is GEIC(V/I)RG (Hu et al., Proc. Natl. Acad. Sci. USA95: 5407-5412, 1998). 32 and 144  94 and 179 Lignin/Tannin Homolog ofcinnamic acid 4-hydroxylase biosynthesis (C4H) isolated from Loliumperenne. The molecules have a conserved cytochrome P450 region in aminoacids 436 to 445 that contains a conserved Cys residue involved in hemebinding (Miles et al., Biochim Biophys Acta 1543: 383-407, 2000). 33 95Lignin/Tannin Homolog of cinnamic acid 4-hydroxylase biosynthesis (C4H)isolated from Festuca arundinacea. The molecule has a conservedCytochrome P450 region in amino acids 440 to 449 that contains aconserved Cys residue involved in heme binding. The cytochrome P450cysteine heme- iron ligand signature consensus is FGxGRRSCPG where theconserved C is the heme iron ligand (Miles et al., Biochim. Biophys.Acta 1543: 383-407, 2000). It also contains an aldehyde dehydrogenasesactive site (Hempel et al., Adv. Exp. Med. Biol. 436: 53-59, 1999) atamino acid residues 428 to 435. A hydrophobic signal peptide region ispresent in amino acid residues 1 to 24. 34 and 145  96 and 180 LigninHomolog of cinnamyl-alcohol dehydrogenase biosynthesis (CAD; EC1.1.1.195) isolated from Lolium perenne. These molecules contain aconserved zinc-containing alcohol dehydrogenase domain (Joernvall etal., Eur. J. Biochem. 167: 195-201, 1987) in amino acid residues 69 to83, with a conserved His residue at position 70. They also contain acytochrome C family heme-binding site signature (Mathews, Prog. Biophys.Mol. Biol. 45: 1-56, 1985) in residues 45 to 50. 35 97 Lignin Homolog ofcinnamyl-alcohol dehydrogenase biosynthesis (CAD; EC 1.1.1.195) isolatedfrom Festuca arundinacea. CAD belongs to the family of zinc-bindingdehydrogenases. This molecule contains a conserved zinc-containingalcohol dehydrogenases domain (Joernvall et al., Eur. J. Biochem. 167:195-201, 1987) in amino acid residues 69 to 83, with a conserved Hisresidue at position 70. It also contains a Cytochrome C familyheme-binding site signature. The cytochrome C family heme-binding sitesignature is CGICHT. In the cytochrome C protein family, the heme groupis covalently attached by thioether bonds to two conserved cysteineresidues. The consensus sequence for this site is Cys-X—X-Cys-His andthe histidine residue is one of the two axial ligands of the heme iron.This arrangement is shared by all proteins known to belong to cytochromeC family (Mathews, Prog. Biophys. Mol. Biol. 45: 1-56, 1985). 36 and 14698 Lignin Homolog of caffeoyl coenzyme A O- biosynthesismethyltransferase (CCoAOMT) (EC 2.1.1.104) isolated from Lolium perenne.37 99 Lignin Homolog of caffeoyl coenzyme A O- biosynthesismethyltransferase (CCoAOMT) (EC 2.1.1.104) isolated from Festucaarundinacea. 38 and 147 100 and 181 Lignin Homolog of cinnamoyl CoA:NADPbiosynthesis oxidoreductase (CCR, EC 1.2.1.44) isolated from Loliumperenne that catalyzes the conversion of cinnamoyl CoA esters to theircorresponding cinnamaldehydes in the first specific step in thesynthesis of the lignin monomers. A hydrophobic region typical of asignal peptide is present in amino acid residues 1 to 24. 39 and 148 101Lignin Homolog of cinnamoyl CoA:NADP biosynthesis oxidoreductase (CCR,EC 1.2.1.44) isolated from Festuca arundinacea that catalyzes theconversion of cinnamoyl CoA esters to their correspondingcinnamaldehydes in the first specific step in the synthesis of thelignin monomers. 40 and 149 102 and 182 Lignin Homolog of caffeic acid3-O-methyltransferase biosynthesis (COMT1) isolated from Festucaarundinacea A conserved consensus phosphopantetheine attachment site wasidentified in amino acid residues 47 to 62. This domain is involved inthe attachment of activated fatty acid and amino-acid groups, with theSer residue at position 52 crucial for the phosphopantetheine attachment(Pugh and Wakil, J. Biol. Chem. 240: 4727-4733, 1965). 41 and 150 103Lignin Homolog of caffeic acid 3-O-methyltransferase biosynthesis(COMT1) isolated from Lolium perenne A conserved consensusphosphopantetheine attachment site was identified in amino acid residues47 to 62. This domain is involved in the attachment of activated fattyacid and amino-acid groups, with the Ser residue at position 52 crucialfor the phosphopantetheine attachment (Pugh and Wakil, J. Biol. Chem.240: 4727-4733, 1965). 42 104 Lignin Homolog of caffeic acid3-O-methyltransferase biosynthesis (COMT1) isolated from Festucaarundinacea A conserved consensus phosphopantetheine attachment site wasidentified in amino acid residues 47 to 62. This domain is involved inthe attachment of activated fatty acid and amino-acid groups, with theSer residue at position 52 crucial for the phosphopantetheine attachment(Pugh and Wakil, J. Biol. Chem. 240: 4727-4733, 1965). 43 105 LigninHomolog of caffeic acid 3-O-methyltransferase biosynthesis (COMT1)isolated from Lolium perenne A conserved consensus phosphopantetheineattachment site was identified in amino acid residues 47 to 62. Thisdomain is involved in the attachment of activated fatty acid andamino-acid groups, with the Ser residue at position 52 crucial for thephosphopantetheine attachment (Pugh and Wakil, J. Biol. Chem. 240:4727-4733, 1965). 44 and 151 106 and 183 Lignin Homolog of ferulate5-hydroxylase (F5H) biosynthesis isolated from Lolium perenne. Themolecules have a conserved cytochrome P450 region in amino acids 463 to472 that contains a conserved Cys residue involved in heme binding(Miles et al., Biochim Biophys Acta 1543: 383-407, 2000). A signalpeptide is present in amino acid residues 1 to 30. 45 107 Lignin Homologof ferulate 5-hydroxylase (F5H) biosynthesis isolated from Festucaarundinaceae. The molecule has a conserved cytochrome P450 region inamino acids 462 to 471 that contains a conserved Cys residue involved inheme binding (Miles et al., Biochim Biophys Acta 1543: 383-407, 2000). Asignal peptide is present in amino acid residues 1 to 30. 46 and 152 108Lignin/Tannin Homolog of phenylalanine ammonia-lyase (EC biosynthesis4.3.1.5) (PAL) isolated from Lolium perenne. The polypeptide has aconserved PAL-histidase region in amino acid residues 193 to 209. 47 and153 109 and 184 Lignin/Tannin Homolog of phenylalanine ammonia-lyase (ECbiosynthesis 4.3.1.5) (PAL) isolated from Festuca arundinacea. Aconserved phenylalanine and histidine ammonia-lyases active sitesignature has been identified in amino acid residues 195 to 210. 48 110Lignin Homolog of peroxidase (PER) isolated from biosynthesis Festucaarundinacea. The conserved peroxidase I region is present in amino acidresidues 188 to 199 and contains a conserved His residue at position 196in the active site, and the conserved peroxidase 2 region is present inamino acid residues 60 to 71, with a conserved His residue at position69 that is involved in heme binding (Kimura and Ikeda- Saito, Proteins3: 113-120, 1988). A signal peptide is present in amino acid residues 1to 27. 49 111 Lignin Homolog of peroxidase (PER) isolated frombiosynthesis Lolium perenne. The conserved peroxidase I region ispresent in amino acid residues 199 to 209 and contains a conserved Hisresidue at position 208 in the active site. A signal peptide is presentin amino acid residues 1 to 33. 50 112 Lignin Homolog of peroxidase(PER) isolated from biosynthesis Festuca arundinacea. The conservedperoxidase I region is present in amino acid residues 180 to 190 andcontains a conserved His residue at position 188 in the active site, andthe conserved peroxidase 2 region is present in amino acid residues 55to 66, with a conserved His residue at position 64 that is involved inheme binding (Kimura and Ikeda- Saito, Proteins 3: 113-120, 1988). Asignal peptide is present in amino acid residues 1 to 22. 51 and 154 113Lignin Homolog of peroxidase (PER) isolated from biosynthesis Loliumperenne. The conserved peroxidase I region is present in amino acidresidues 199 to 209 and contains a conserved His residue at position 207in the active site, and the conserved peroxidase 2 region is present inamino acid residues 70 to 80, with a conserved His residue at position78 that is involved in heme binding (Kimura and Ikeda-Saito, Proteins 3:113-120, 1988). A signal peptide is present in amino acid residues 1 to20. 52 and 155 114 Lignin Homolog of peroxidase (PER) isolated frombiosynthesis Lolium perenne. The conserved peroxidase I region ispresent in amino acid residues 198 to 208 and contains a conserved Hisresidue at position 206 in the active site (Kimura and Ikeda-Saito,Proteins 3: 113-120, 1988). A signal peptide is present in amino acidresidues 1 to 34. 53, 156, and 115, 185, an Lignin Homolog of peroxidase(PER) isolated from 162 190 biosynthesis Lolium perenne. The conservedperoxidase I region is present in amino acid residues 157 to 168, 188 to199, and 190 to 201, respectively and contain a conserved His residue atposition 165, 196 and 198, respectively in the active site, and theconserved peroxidase 2 region is present in amino acid residues 29 to41, 60 to 72 and 62 to 74, respectively, with a conserved His residue atposition 38, 69 and 71, respectively that is involved in heme binding(Kimura and Ikeda-Saito, Proteins 3: 113-120, 1988). 54 116 LigninHomolog of peroxidase (PER) isolated from biosynthesis Festucaarundinacea. The conserved peroxidase I region is present in amino acidresidues 176 to 186 and contains a conserved His residue at position 184in the active site, and the conserved peroxidase 2 region is present inamino acid residues 55 to 67, with a conserved His residue at position64 that is involved in heme binding (Kimura and Ikeda- Saito, Proteins3: 113-120, 1988). A signal peptide is present in amino acid residues 1to 22. 55 117 Tannin Homolog of chalcone isomerase (CHI) isolatedBiosynthesis from Lolium perenne. The molecule contains a chalconeisomerase region at amino acid residues 1 to 213. 56 118 Tannin Homologof chalcone isomerase (CHI). The Biosynthesis molecule contains achalcone isomerase region at amino acid residues 23 to 235. 57 and 157119 and 186 Tannin Homolog of Chalcone Synthase (CHS) isolatedBiosynthesis from Lolium perenne and that is an important enzyme inflavonoid synthesis. The molecules contain a conserved chalcone synthaseactive site (Lanz et al., J. Biol. Chem. 266: 9971-9976, 1991) at aminoacid residues 166 to 175, with the conserved Cys residue at position167. 58 and 158 120 and 187 Tannin Homolog ofdihydroflavonal-4-reductase Biosynthesis (DFR) isolated from Festucaarundinacea. 59 and 159 121 and 188 Tannin Homolog ofdihydroflavonal-4-reductase Biosynthesis (DFR) isolated from Loliumperenne. 60 and 160 122 and 189 Tannin Homolog ofdihydroflavonal-4-reductase Biosynthesis (DFR) isolated from Loliumperenne. These molecules contain a conserved ATP/GTP binding site atamino acid residues 84 to 91 and 86 to 93, respectively, known as the“A” sequence (Walker et al., EMBO J. 1: 945-951, 1982) or “P-loop”(Saraste et al., Trends Biochem. Sci. 15: 430-434, 1990). 61 and 161 123Tannin Homolog of flavanone 3-βhydroxylase (F3βH) biosynthesis isolatedfrom Lolium perenne. 62 124 Tannin Homolog of flavanone 3-βhydroxylase(F3βH) Biosynthesis isolated from Festuca arundinacea.

All the polynucleotides and polypeptides provided by the presentinvention are isolated and purified, as those terms are commonly used inthe art. Preferably, the polypeptides and polynucleotides are at leastabout 80% pure, more preferably at least about 90% pure, and mostpreferably at least about 99% pure.

The word “polynucleotide(s),” as used herein, means a polymericcollection of nucleotides, and includes DNA and corresponding RNAmolecules and both single and double stranded molecules, including RNAi,HnRNA and mRNA molecules, sense and anti-sense strands of DNA and RNAmolecules, and comprehends cDNA, genomic DNA, and wholly or partiallysynthesized polynucleotides. A polynucleotide of the present inventionmay be an entire gene, or any portion thereof. As used herein, a “gene”is a DNA sequence which codes for a functional protein or RNA molecule.Operable anti-sense polynucleotides may comprise a fragment of thecorresponding polynucleotide, and the definition of “polynucleotide”therefore includes all operable anti-sense fragments. Anti-sensepolynucleotides and techniques involving anti-sense polynucleotides arewell known in the art and are described, for example, in Robinson-Benionet al., Methods in Enzymol. 254(23): 363-375, 1995 and Kawasaki et al.,Artific. Organs 20(8): 836-848, 1996.

In specific embodiments, the present invention provides isolatedpolynucleotides comprising a sequence of SEQ ID NO: 1-62 and 125-162;polynucleotides comprising variants of SEQ ID NO: 1-62 and 125-162;polynucleotides comprising extended sequences of SEQ ID NO: 1-62 and125-162 and their variants, oligonucleotide primers and probescorresponding to the sequences set out in SEQ ID NO: 1-62 and 125-162and their variants, polynucleotides comprising at least a specifiednumber of contiguous residues of any of SEQ ID NO: 1-62 and 125-162(x-mers), and polynucleotides comprising extended sequences whichinclude portions of the sequences set out in SEQ ID NO: 1-62 and125-162, all of which are referred to herein, collectively, as“polynucleotides of the present invention.” Polynucleotides thatcomprise complements of such polynucleotide sequences, reversecomplements of such polynucleotide sequences, or reverse sequences ofsuch polynucleotide sequences, together with variants of such sequences,are also provided.

The definition of the terms “complement(s),” “reverse complement(s),”and “reverse sequence(s),” as used herein, is best illustrated by thefollowing example. For the sequence 5′ AGGACC 3′, the complement,reverse complement, and reverse sequence are as follows:

complement 3′ TCCTGG 5′ reverse complement 3′ GGTCCT 5′ reverse sequence5′ CCAGGA 3′.

Preferably, sequences that are complements of a specifically recitedpolynucleotide sequence are complementary over the entire length of thespecific polynucleotide sequence.

As used herein, the term “x-mer,” with reference to a specific value of“x,” refers to a polynucleotide comprising at least a specified number(“x”) of contiguous residues of: any of the polynucleotides provided inSEQ ID NO: 1-62 and 125-162. The value of x may be from about 20 toabout 600, depending upon the specific sequence.

Polynucleotides of the present invention comprehend polynucleotidescomprising at least a specified number of contiguous residues (x-mers)of any of the polynucleotides identified as SEQ ID NO: 1-62 and 125-162,or their variants. Similarly, polypeptides of the present inventioncomprehend polypeptides comprising at least a specified number ofcontiguous residues (x-mers) of any of the polypeptides identified asSEQ ID NO: 63-124 and 163-190. According to preferred embodiments, thevalue of x is at least 20, more preferably at least 40, more preferablyyet at least 60, and most preferably at least 80. Thus, polynucleotidesof the present invention include polynucleotides comprising a 20-mer, a40-mer, a 60-mer, an 80-mer, a 100-mer, a 120-mer, a 150-mer, a 180-mer,a 220-mer, a 250-mer; or a 300-mer, 400-mer, 500-mer or 600-mer of apolynucleotide provided in SEQ ID NO: 1-62 and 125-162, or a variant ofone of the polynucleotides corresponding to the polynucleotides providedin SEQ ID NO: 1-62 and 125-162. Polypeptides of the present inventioninclude polypeptides comprising a 20-mer, a 40-mer, a 60-mer, an 80-mer,a 100-mer, a 120-mer, a 150-mer, a 180-mer, a 220-mer, a 250-mer; or a300-mer, 400-mer, 500-mer or 600-mer of a polypeptide provided in SEQ IDNO: 63-124 and 163-190, or a variant thereof.

Polynucleotides of the present invention were isolated by highthroughput sequencing of cDNA libraries comprising forage grass tissuecollected from Lolium perenne and Festuca arundinacea. Some of thepolynucleotides of the present invention may be “partial” sequences, inthat they do not represent a full-length gene encoding a full-lengthpolypeptide. Such partial sequences may be extended by analyzing andsequencing various DNA libraries using primers and/or probes and wellknown hybridization and/or PCR techniques. Partial sequences may beextended until an open reading frame encoding a polypeptide, afull-length polynucleotide and/or gene capable of expressing apolypeptide, or another useful portion of the genome is identified. Suchextended sequences, including full-length polynucleotides and genes, aredescribed as “corresponding to” a sequence identified as one of thesequences of SEQ ID NO: 1-62 and 125-162 or a variant thereof, or aportion of one of the sequences of SEQ ID NO: 1-62 and 125-162 or avariant thereof, when the extended polynucleotide comprises anidentified sequence or its variant, or an identified contiguous portion(x-mer) of one of the sequences of SEQ ID NO: 1-62 and 125-162 or avariant thereof. Similarly, RNA sequences, reverse sequences,complementary sequences, anti-sense sequences and the like,corresponding to the polynucleotides of the present invention, may beroutinely ascertained and obtained using the cDNA sequences identifiedas SEQ ID NO: 1-62 and 125-162.

The polynucleotides identified as SEQ ID NO: 1-62 and 125-162 containopen reading frames (“ORFs”) or partial open reading frames encodingpolypeptides and functional portions of polypeptides. Additionally, openreading frames encoding polypeptides may be identified in extended orfull length sequences corresponding to the sequences set out as SEQ IDNO: 1-62 and 125-162. Open reading frames may be identified usingtechniques that are well known in the art. These techniques include, forexample, analysis for the location of known start and stop codons, mostlikely reading frame identification based on codon frequencies, etc.These techniques include, for example, analysis for the location ofknown start and stop codons, most likely reading frame identificationbased on codon frequencies, etc. Suitable tools and software for ORFanalysis are well known in the art and include, for example, GeneWise,available from The Sanger Center, Wellcome Trust Genome Campus, Hinxton,Cambridge, CB10 1SA, United Kingdom; Diogenes, available fromComputational Biology Centers, University of Minnesota, Academic HealthCenter, UMHG Box 43 Minneapolis Minn. 55455; and GRAIL, available fromthe Informatics Group, Oak Ridge National Laboratories, Oak Ridge, Tenn.Once a partial open reading frame is identified, the polynucleotide maybe extended in the area of the partial open reading frame usingtechniques that are well known in the art until the polynucleotide forthe full open reading frame is identified.

Once open reading frames are identified in the polynucleotides of thepresent invention, the open reading frames may be isolated and/orsynthesized. Expressible genetic constructs comprising the open readingframes and suitable promoters, initiators, terminators, etc., which arewell known in the art, may then be constructed. Such genetic constructsmay be introduced into a host cell to express the polypeptide encoded bythe open reading frame. Suitable host cells may include variousprokaryotic and eukaryotic cells, including plant cells, mammaliancells, bacterial cells, algae and the like.

The polynucleotides of the present invention may be isolated by highthroughput sequencing of cDNA libraries prepared from forage grasstissue, as described below in Example 1. Alternatively, oligonucleotideprobes and primers based on the sequences provided in SEQ ID NO: 1-62and 125-162 can be synthesized as detailed below, and used to identifypositive clones in either cDNA or genomic DNA libraries from foragegrass tissue cells by means of hybridization or polymerase chainreaction (PCR) techniques. Hybridization and PCR techniques suitable foruse with such oligonucleotide probes are well known in the art (see, forexample, Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51:263,1987; Erlich, ed., PCR technology, Stockton Press: NY, 1989; andSambrook et al., eds., Molecular cloning: a laboratory manual, 2nd ed.,CSHL Press: Cold Spring Harbor, N.Y., 1989). In addition to DNA-DNAhybridization, DNA-RNA or RNA-RNA hybridization assays are alsopossible. In the first case, the mRNA from expressed genes would then bedetected instead of genomic DNA or cDNA derived from mRNA of the sample.In the second case, RNA probes could be used. Artificial analogs of DNAhybridizing specifically to target sequences could also be employed.Positive clones may be analyzed by restriction enzyme digestion, DNAsequencing or the like.

The polynucleotides of the present invention may also, or alternatively,be synthesized using techniques that are well known in the art. Thepolynucleotides may be synthesized, for example, using automatedoligonucleotide synthesizers (e.g., Beckman Oligo 1000M DNA Synthesizer;Beckman Coulter Ltd., Fullerton, Calif.) to obtain polynucleotidesegments of up to 50 or more nucleic acids. A plurality of suchpolynucleotide segments may then be ligated using standard DNAmanipulation techniques that are well known in the art of molecularbiology. One conventional and exemplary polynucleotide synthesistechnique involves synthesis of a single stranded polynucleotide segmenthaving, for example, 80 nucleic acids, and hybridizing that segment to asynthesized complementary 85 nucleic acid segment to produce a 5nucleotide overhang. The next segment may then be synthesized in asimilar fashion, with a 5 nucleotide overhang on the opposite strand.The “sticky” ends ensure proper ligation when the two portions arehybridized. In this way, a complete polynucleotide of the presentinvention may be synthesized entirely in vitro.

Oligonucleotide probes and primers complementary to and/or correspondingto SEQ ID NO: 1-62 and 125-162, and variants of those sequences, arealso comprehended by the present invention. Such oligonucleotide probesand primers are substantially complementary to the polynucleotide ofinterest over a certain portion of the polynucleotide. Anoligonucleotide probe or primer is described as “corresponding to” apolynucleotide of the present invention, including one of the sequencesset out as SEQ ID NO: 1-62 and 125-162 or a variant thereof, if theoligonucleotide probe or primer, or its complement, is contained withinone of the sequences set out as SEQ ID NO: 1-62 and 125-162 or a variantof one of the specified sequences.

Two single stranded sequences are said to be substantially complementarywhen the nucleotides of one strand, optimally aligned and compared, withthe appropriate nucleotide insertions and/or deletions, pair with atleast 80%, preferably at least 90% to 95%, and more preferably at least98% to 100%, of the nucleotides of the other strand. Alternatively,substantial complementarity exists when a first DNA strand willselectively hybridize to a second DNA strand under stringenthybridization conditions.

In specific embodiments, the oligonucleotide probes and/or primerscomprise at least about 6 contiguous residues, more preferably at leastabout 10 contiguous residues, and most preferably at least about 20contiguous residues complementary to a polynucleotide sequence of thepresent invention. Probes and primers of the present invention may befrom about 8 to 100 base pairs in length, preferably from about 10 to 50base pairs in length, and more preferably from about 15 to 40 base pairsin length. The probes can be easily selected using procedures well knownin the art, taking into account DNA-DNA hybridization stringencies,annealing and melting temperatures, potential for formation of loops,and other factors which are well known in the art. Preferred techniquesfor designing PCR primers are disclosed in Dieffenbach and Dyksler, PCRPrimer: a laboratory manual, CSHL Press: Cold Spring Harbor, N.Y., 1995.A software program suitable for designing probes, and especially fordesigning PCR primers, is available from Premier Biosoft International,3786 Corina Way, Palo Alto, Calif. 94303-4504.

The isolated polynucleotides of the present invention also have utilityin genome mapping, in physical mapping, and in positional cloning ofgenes.

The polynucleotides identified as SEQ ID NO: 1-62 and 125-162 wereisolated from cDNA clones and represent sequences that are expressed inthe tissue from which the cDNA was prepared. RNA sequences, reversesequences, complementary sequences, anti-sense sequences, and the like,corresponding to the polynucleotides of the present invention, may beroutinely ascertained and obtained using the cDNA sequences identifiedas SEQ ID NO: 1-62 and 125-162.

Identification of genomic DNA and heterologous species DNA can beaccomplished by standard DNA/DNA hybridization techniques, underappropriately stringent conditions, using all or part of apolynucleotide sequence as a probe to screen an appropriate library.Alternatively, PCR techniques using oligonucleotide primers that aredesigned based on known genomic DNA, cDNA and protein sequences can beused to amplify and identify genomic and cDNA sequences.

In another aspect, the present invention provides isolated polypeptidesencoded by the above polynucleotides. As used herein, the term“polypeptide” encompasses amino acid chains of any length, includingfull-length proteins, wherein the amino acid residues are linked bycovalent peptide bonds. The term “polypeptide encoded by apolynucleotide” as used herein, includes polypeptides encoded by apolynucleotide that comprises a partial isolated polynucleotide sequenceprovided herein. In specific embodiments, the inventive polypeptidescomprise an amino acid sequence selected from the group consisting ofSEQ ID NO: 63-124 and 163-190, as well as variants of such sequences.

As noted above, polypeptides of the present invention may be producedrecombinantly by inserting a polynucleotide sequence of the presentinvention encoding the polypeptide into an expression vector andexpressing the polypeptide in an appropriate host. Any of a variety ofexpression vectors known to those of ordinary skill in the art may beemployed. Expression may be achieved in any appropriate host cell thathas been transformed or transfected with an expression vector containinga polynucleotide molecule that encodes a recombinant polypeptide.Suitable host cells include prokaryotes, yeast, and higher eukaryoticcells. Preferably, the host cells employed are plant, E. coli, insect,yeast, or a mammalian cell line such as COS or 293T. The polynucleotidesequences expressed in this manner may encode naturally occurringpolypeptides, portions of naturally occurring polypeptides, or othervariants thereof. The expressed polypeptides may be used in variousassays known in the art to determine their biological activity. Suchpolypeptides may also be used to raise antibodies, to isolatecorresponding interacting proteins or other compounds, and toquantitatively determine levels of interacting proteins or othercompounds.

In a related aspect, polypeptides are provided that comprise at least afunctional portion of a polypeptide having an amino acid sequenceselected from the group consisting of sequences provided in SEQ ID NO:63-124 and 163-190, and variants thereof. As used herein, the“functional portion” of a polypeptide is that portion which contains anactive site essential for affecting the function of the polypeptide, forexample, a portion of the molecule that is capable of binding one ormore reactants. The active site may be made up of separate portionspresent on one or more polypeptide chains and will generally exhibithigh binding affinity. Functional portions of a polypeptide may beidentified by first preparing fragments of the polypeptide by eitherchemical or enzymatic digestion of the polypeptide, or by mutationanalysis of the polynucleotide that encodes the polypeptide andsubsequent expression of the resulting mutant polypeptides. Thepolypeptide fragments or mutant polypeptides are then tested todetermine which portions retain biological activity, using methods wellknown to those of skill in the art, including the representative assaysdescribed below.

Portions and other variants of the inventive polypeptides may begenerated by synthetic or recombinant means. Synthetic polypeptideshaving fewer than about 100 amino acids, and generally fewer than about50 amino acids, may be generated using techniques well known to those ofordinary skill in the art. For example, such polypeptides may besynthesized using any of the commercially available solid-phasetechniques, such as the Merrifield solid-phase synthesis method, whereamino acids are sequentially added to a growing amino acid chain. SeeMerrifield, J. Am. Chem. Soc. 85:2149-2146, 1963. Equipment forautomated synthesis of polypeptides is commercially available fromsuppliers such as Perkin Elmer/Applied Biosystems, Inc. (Foster City,Calif.), and may be operated according to the manufacturer'sinstructions. Variants of a native polypeptide may be prepared usingstandard mutagenesis techniques, such as oligonucleotide-directedsite-specific mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA82:488-492, 1985). Sections of DNA sequences may also be removed usingstandard techniques to permit preparation of truncated polypeptides.

As used herein, the term “variant” comprehends nucleotide or amino acidsequences different from the specifically identified sequences, whereinone or more nucleotides or amino acid residues is deleted, substituted,or added. Variants may be naturally occurring allelic variants, ornon-naturally occurring variants. Variant sequences (polynucleotide orpolypeptide) preferably exhibit at least 75%, more preferably at least80%, more preferably at least 90%, more preferably yet at least 95% andmost preferably, at least 98% identity to a sequence of the presentinvention. The percentage identity is determined by aligning the twosequences to be compared as described below, determining the number ofidentical residues in the aligned portion, dividing that number by thetotal number of residues in the inventive (queried) sequence, andmultiplying the result by 100.

Polynucleotides and polypeptides having a specified percentage identityto a polynucleotide or polypeptide identified in one of SEQ ID NO: 1-190thus share a high degree of similarity in their primary structure. Inaddition to a specified percentage identity to a polynucleotide orpolypeptide of the present invention, variant polynucleotides andpolypeptides preferably have additional structural and/or functionalfeatures in common with a polynucleotide of the present invention.Polynucleotides having a specified degree of identity to, or capable ofhybridizing to, a polynucleotide of the present invention preferablyadditionally have at least one of the following features: (1) theycontain an open reading frame, or partial open reading frame, encoding apolypeptide, or a functional portion of a polypeptide, havingsubstantially the same functional properties as the polypeptide, orfunctional portion thereof, encoded by a polynucleotide in the recitedSEQ ID NO:; or (2) they contain identifiable domains in common.Similarly, polypeptides having a specified degree of identity to apolypeptide of the present invention preferably additionally have atleast one of the following features: (1) they have substantially thesame functional properties as the polypeptide in the recited SEQ ID NO:;or (2) they contain identifiable domains in common.

Polynucleotide or polypeptide sequences may be aligned, and percentagesof identical nucleotides or amino acids in a specified region may bedetermined against another polynucleotide or polypeptide, using computeralgorithms that are publicly available. The BLASTN and FASTA algorithms,set to the default parameters described in the documentation anddistributed with the algorithm, may be used for aligning and identifyingthe similarity of polynucleotide sequences. The alignment and similarityof polypeptide sequences may be examined using the BLASTP algorithm.BLASTX and FASTX algorithms compare nucleotide query sequencestranslated in all reading frames against polypeptide sequences. TheFASTA and FASTX algorithms are described in Pearson and Lipman, Proc.Natl. Acad. Sci. USA 85:2444-2448, 1988; and in Pearson, Methods inEnzymol. 183:63-98, 1990. The FASTA software package is available fromthe University of Virginia by contacting the Assistant Provost forResearch, University of Virginia, PO Box 9025, Charlottesville, Va.22906-9025. The BLASTN software is available from the National Centerfor Biotechnology Information (NCBI), National Library of Medicine,Building 38A, Room 8N805, Bethesda, Md. 20894. The BLASTN algorithmVersion 2.0.11 [Jan. 20, 2000] and Version 2.2.1 [Apr. 13, 2001] set tothe default parameters described in the documentation and distributedwith the algorithm, are preferred for use in the determination ofpolynucleotide variants according to the present invention. The use ofthe BLAST family of algorithms, including BLASTN, BLASTP and BLASTX, isdescribed in the publication of Altschul et al., “Gapped BLAST andPSI-BLAST: a new generation of protein database search programs,”Nucleic Acids Res. 25:3389-3402, 1997.

The following running parameters are preferred for determination ofalignments and similarities using BLASTN that contribute to the E valuesand percentage identity for polynucleotides: Unix running command withthe following default parameters: blastall -p blastn -d embldb -e 10 -G0 -E 0 -r 1 -v 30 -b 30 -i queryseq -o results; and parameters are: -pProgram Name [String]; -d Database [String]; -e Expectation value (E)[Real]; -G Cost to open a gap (zero invokes default behavior) [Integer];-E Cost to extend a gap (zero invokes default behavior) [Integer]; -rReward for a nucleotide match (BLASTN only) [Integer]; -v Number ofone-line descriptions (V) [Integer]; -b Number of alignments to show (B)[Integer]; -i Query File [File In]; -o BLAST report Output File [FileOut] Optional.

The following running parameters are preferred for determination ofalignments and similarities using BLASTP that contribute to the E valuesand percentage identity of polypeptide sequences: blastall -p blastp -dswissprotdb -e 10 -G 0 -E 0 -v 30 -b 30 -i queryseq -o results; theparameters are: -p Program Name [String]; -d Database [String]; -eExpectation value (E) [Real]; -G Cost to open a gap (zero invokesdefault behavior) [Integer]; -E Cost to extend a gap (zero invokesdefault behavior) [Integer]; -v Number of one-line descriptions (v)[Integer]; -b Number of alignments to show (b) [Integer]; -I Query File[File In]; -o BLAST report Output File [File Out] Optional.

The “hits” to one or more database sequences by a queried sequenceproduced by BLASTN, BLASTP, FASTA, or a similar algorithm, align andidentify similar portions of sequences. The hits are arranged in orderof the degree of similarity and the length of sequence overlap. Hits toa database sequence generally represent an overlap over only a fractionof the sequence length of the queried sequence.

As noted above, the percentage identity of a polynucleotide orpolypeptide sequence is determined by aligning polynucleotide andpolypeptide sequences using appropriate algorithms, such as BLASTN orBLASTP, respectively, set to default parameters; identifying the numberof identical nucleic or amino acids over the aligned portions; dividingthe number of identical nucleic or amino acids by the total number ofnucleic or amino acids of the polynucleotide or polypeptide of thepresent invention; and then multiplying by 100 to determine thepercentage identity. By way of example, a queried polynucleotide having220 nucleic acids has a hit to a polynucleotide sequence in the EMBLdatabase having 520 nucleic acids over a stretch of 23 nucleotides inthe alignment produced by the BLASTN algorithm using the defaultparameters. The 23-nucleotide hit includes 21 identical nucleotides, onegap and one different nucleotide. The percentage identity of the queriedpolynucleotide to the hit in the EMBL database is thus 21/220 times 100,or 9.5%. The percentage identity of polypeptide sequences may bedetermined in a similar fashion.

The BLASTN and BLASTX algorithms also produce “Expect” values forpolynucleotide and polypeptide alignments. The Expect value (E)indicates the number of hits one can “expect” to see over a certainnumber of contiguous sequences by chance when searching a database of acertain size. The Expect value is used as a significance threshold fordetermining whether the hit to a database indicates true similarity. Forexample, an E value of 0.1 assigned to a polynucleotide hit isinterpreted as meaning that in a database of the size of the EMBLdatabase, one might expect to see 0.1 matches over the aligned portionof the sequence with a similar score simply by chance. By thiscriterion, the aligned and matched portions of the sequences then have aprobability of 90% of being related. For sequences having an E value of0.01 or less over aligned and matched portions, the probability offinding a match by chance in the EMBL database is 1% or less using theBLASTN algorithm. E values for polypeptide sequences may be determinedin a similar fashion using various polypeptide databases, such as theSwissProt database.

According to one embodiment, “variant” polynucleotides and polypeptides,with reference to each of the polynucleotides and polypeptides of thepresent invention, preferably comprise sequences having the same numberor fewer nucleotides or amino acids than each of the polynucleotides orpolypeptides of the present invention and producing an E value of 0.01or less when compared to the polynucleotide or polypeptide of thepresent invention. That is, a variant polynucleotide or polypeptide isany sequence that has at least a 99% probability of being related to thepolynucleotide or polypeptide of the present invention, measured ashaving an E value of 0.01 or less using the BLASTN or BLASTX algorithmsset at the default parameters. According to a preferred embodiment, avariant polynucleotide is a sequence having the same number or fewernucleic acids than a polynucleotide of the present invention that has atleast a 99% probability of being related to the polynucleotide of thepresent invention, measured as having an E value of 0.01 or less usingthe BLASTN algorithm set at the default parameters. Similarly, accordingto a preferred embodiment, a variant polypeptide is a sequence havingthe same number or fewer amino acids than a polypeptide of the presentinvention that has at least a 99% probability of being related as thepolypeptide of the present invention, measured as having an E value of0.01 or less using the BLASTP algorithm set at the default parameters.

In an alternative embodiment, variant polynucleotides are sequences thathybridize to a polynucleotide of the present invention under stringentconditions. Stringent hybridization conditions for determiningcomplementarity include salt conditions of less than about 1 M, moreusually less than about 500 mM, and preferably less than about 200 mM.Hybridization temperatures can be as low as 5° C., but are generallygreater than about 22° C., more preferably greater than about 30° C.,and most preferably greater than about 37° C. Longer DNA fragments mayrequire higher hybridization temperatures for specific hybridization.Since the stringency of hybridization may be affected by other factorssuch as probe composition, presence of organic solvents, and extent ofbase mismatching, the combination of parameters is more important thanthe absolute measure of any one alone. An example of “stringentconditions” is prewashing in a solution of 6×SSC, 0.2% SDS; hybridizingat 65° C., 6×SSC, 0.2% SDS overnight; followed by two washes of 30minutes each in 1×SSC, 0.1% SDS at 65° C. and two washes of 30 minuteseach in 0.2×SSC, 0.1% SDS at 65° C.

The present invention also encompasses polynucleotides that differ fromthe disclosed sequences but that, as a consequence of the discrepancy ofthe genetic code, encode a polypeptide having similar enzymatic activityto a polypeptide encoded by a polynucleotide of the present invention.Thus, polynucleotides comprising sequences that differ from thepolynucleotide sequences recited in SEQ ID NO: 1-62 and 125-162, orcomplements, reverse sequences, or reverse complements of thosesequences, as a result of conservative substitutions are contemplated byand encompassed within the present invention. Additionally,polynucleotides comprising sequences that differ from the polynucleotidesequences recited in SEQ ID NO: 1-62 and 125-162, or complements,reverse complements or reverse sequences thereof, as a result ofdeletions and/or insertions totaling less than 10% of the total sequencelength are also contemplated by and encompassed within the presentinvention. Similarly, polypeptides comprising sequences that differ fromthe polypeptide sequences recited in SEQ ID NO: 63-124 and 163-190 as aresult of amino acid substitutions, insertions, and/or deletionstotaling less than 10% of the total sequence length are contemplated byand encompassed within the present invention, provided the variantpolypeptide has activity in a lignin, fructan or tannin biosyntheticpathway.

In another aspect, the present invention provides genetic constructscomprising, in the 5′-3′ direction, a gene promoter sequence; an openreading frame coding for at least a functional portion of a polypeptideof the present invention; and a gene termination sequence. The openreading frame may be orientated in either a sense or anti-sensedirection. For applications where amplification of lignin, fructan ortannin synthesis is desired, the open reading frame may be inserted inthe construct in a sense orientation, such that transformation of atarget organism with the construct will lead to an increase in thenumber of copies of the gene and therefore an increase in the amount ofenzyme. When down-regulation of lignin, fructan or tannin synthesis isdesired, the open reading frame may be inserted in the construct in ananti-sense orientation, such that the RNA produced by transcription ofthe polynucleotide is complementary to the endogenous mRNA sequence.This, in turn, will result in a decrease in the number of copies of thegene and therefore a decrease in the amount of enzyme. Alternatively,regulation may be achieved by inserting appropriate sequences orsubsequences (e.g., DNA or RNA) in ribozyme constructs.

Genetic constructs comprising a non-coding region of a gene coding for apolypeptide of the present invention, or a nucleotide sequencecomplementary to a non-coding region, together with a gene promotersequence and a gene termination sequence, are also provided. As usedherein the term “non-coding region” includes both transcribed sequenceswhich are not translated, and non-transcribed sequences within about2000 base pairs 5′ or 3′ of the translated sequences or open readingframes. Examples of non-coding regions which may be usefully employed inthe inventive constructs include introns and 5′-non-coding leadersequences. Transformation of a target plant with such a geneticconstruct may lead to a reduction in the amount of lignin, fructan ortannin synthesized by the plant by the process of cosuppression, in amanner similar to that discussed, for example, by Napoli et al., PlantCell 2:279-290, 1990; and de Carvalho Niebel et al., Plant Cell7:347-358, 1995.

The genetic constructs of the present invention further comprise a genepromoter sequence and a gene termination sequence, operably linked tothe polynucleotide to be transcribed, which control expression of thegene. The gene promoter sequence is generally positioned at the 5′ endof the polynucleotide to be transcribed, and is employed to initiatetranscription of the polynucleotide. Gene promoter sequences aregenerally found in the 5′ non-coding region of a gene but they may existin introns (Luehrsen, Mol. Gen. Genet. 225:81-93, 1991). When theconstruct includes an open reading frame in a sense orientation, thegene promoter sequence also initiates translation of the open readingframe. For genetic constructs comprising either an open reading frame inan anti-sense orientation or a non-coding region, the gene promotersequence consists only of a transcription initiation site having a RNApolymerase binding site.

A variety of gene promoter sequences which may be usefully employed inthe genetic constructs of the present invention are well known in theart. The promoter gene sequence, and also the gene termination sequence,may be endogenous to the target plant host or may be exogenous, providedthe promoter is functional in the target host. For example, the promoterand termination sequences may be from other plant species, plantviruses, bacterial plasmids and the like. Preferably, gene promoter andtermination sequences are from the inventive sequences themselves.

Factors influencing the choice of promoter include the desired tissuespecificity of the construct, and the timing of transcription andtranslation. For example, constitutive promoters, such as the 35SCauliflower Mosaic Virus (CaMV 35S) promoter, will affect the activityof the enzyme in all parts of the plant. Use of a tissue specificpromoter will result in production of the desired sense or anti-senseRNA only in the tissue of interest. With DNA constructs employinginducible gene promoter sequences, the rate of RNA polymerase bindingand initiation can be modulated by external stimuli, such as light,heat, anaerobic stress, alteration in nutrient conditions and the like.Temporally regulated promoters can be employed to effect modulation ofthe rate of RNA polymerase binding and initiation at a specific timeduring development of a transformed cell. Preferably, the originalpromoters from the enzyme gene in question, or promoters from a specifictissue-targeted gene in the organism to be transformed, such as Loliumor Festuca, are used. Grass promoters different from the original genemay also be usefully employed in the inventive genetic constructs inorder to prevent feedback inhibition. For example, thefructosyltransferase gene will be regulated by sucrose sensing systems;therefore removing the gene from under control of its normal promoterallows the gene to be active all the time. Other examples of genepromoters which may be usefully employed in the present inventioninclude, mannopine synthase (mas), octopine synthase (ocs) and thosereviewed by Chua et al., Science 244:174-181, 1989.

The gene termination sequence, which is located 3′ to the polynucleotideto be transcribed, may come from the same gene as the gene promotersequence or may be from a different gene. Many gene terminationsequences known in the art may be usefully employed in the presentinvention, such as the 3′ end of the Agrobacterium tumefaciens nopalinesynthase gene. However, preferred gene terminator sequences are thosefrom the original enzyme gene or from the target species to betransformed.

The genetic constructs of the present invention may also contain aselection marker that is effective in plant cells, to allow for thedetection of transformed cells containing the inventive construct. Suchmarkers, which are well known in the art, typically confer resistance toone or more toxins. One example of such a marker is the NPTII gene whoseexpression results in resistance to kanamycin or hygromycin, antibioticswhich are usually toxic to plant cells at a moderate concentration(Rogers et al., in Weissbach A and H, eds., Methods for Plant MolecularBiology, Academic Press Inc.: San Diego, Calif., 1988). Alternatively,the presence of the desired construct in transformed cells can bedetermined by means of other techniques well known in the art, such asSouthern and Western blots.

Techniques for operatively linking the components of the inventivegenetic constructs are well known in the art and include the use ofsynthetic linkers containing one or more restriction endonuclease sitesas described, for example, by Sambrook et al., (Molecular cloning: alaboratory manual, CSHL Press: Cold Spring Harbor, N.Y., 1989). Thegenetic construct of the present invention may be linked to a vectorhaving at least one replication system, for example, E. coli, wherebyafter each manipulation, the resulting construct can be cloned andsequenced and the correctness of the manipulation determined.

The genetic constructs of the present invention may be used to transforma variety of plants, both monocotyledonous (e.g., grasses, maize/corn,grains, oats, rice, sorghum, millet, rye, sugar cane, wheat and barley),dicotyledonous (e.g., Arabidopsis, tobacco, legumes, alfalfa, oaks,eucalyptus, maple), and gymnosperms. In a preferred embodiment, theinventive genetic constructs are employed to transform grasses.Preferably the target plant is selected from the group consisting ofLolium and Festuca species, most preferably from the group consisting ofLolium perenne and Festuca arundinacea. Other plants that may beusefully transformed with the inventive genetic constructs include otherspecies of ryegrass and fescue, including, but not limited to Loliummultiflorum (Italian ryegrass), Lolium hybridum (hybrid ryegrass),Lolium rigidum (Wimerra grass), Lolium temulentum (darnel), Festucarubra (red fescue) and Festuca pratensis (meadow fescue). As discussedabove, transformation of a plant with a genetic construct of the presentinvention will produce a modified lignin, fructan or tannin content inthe plant.

The production of RNA in target cells may be controlled by choice of thepromoter sequence, or by selecting the number of functional copies orthe site of integration of the polynucleotides incorporated into thegenome of the target organism. A target plant may be transformed withmore than one construct of the present invention, thereby modulating thelignin, fructan and/or tannin biosynthetic pathways by affecting theactivity of more than one enzyme, affecting enzyme activity in more thanone tissue or affecting enzyme activity at more than one expressiontime. Similarly, a construct may be assembled containing more than oneopen reading frame coding for an enzyme encoded by a polynucleotide ofthe present invention or more than one non-coding region of a genecoding for such an enzyme. The polynucleotides of the present inventionmay also be employed in combination with other known sequences encodingenzymes involved in the lignin, fructan and/or tannin biosyntheticpathways. In this manner, more than one biosynthetic pathway may bemodulated, or a lignin, fructan or tannin biosynthetic pathway may beadded to a plant to produce a plant having an altered phenotype.

Techniques for stably incorporating genetic constructs into the genomeof target plants are well known in the art and include Agrobacteriumtumefaciens mediated introduction, electroporation, protoplast fusion,injection into reproductive organs, injection into immature embryos,high velocity projectile introduction and the like. The choice oftechnique will depend upon the target plant to be transformed. Forexample, dicotyledonous plants and certain monocots and gymnosperms maybe transformed by Agrobacterium Ti plasmid technology, as described, forexample by Bevan, Nucleic Acid Res. 12:8711-8721, 1984. Targets for theintroduction of the genetic constructs of the present invention includetissues, such as leaf tissue, disseminated cells, protoplasts, seeds,embryos, meristematic regions; cotyledons, hypocotyls, and the like.Transformation techniques which may be usefully employed in theinventive methods include those taught by Ellis et al., Plant CellReports, 8:16-20, 1989; Wilson et al., Plant Cell Reports 7:704-707,1989; Tautorus et al., Theor. Appl. Genet. 78:531-536, 198; Hiei et al.,Plant J. 6:271-282, 1994; and Ishida et al., Nature Biotechnol.14:745-750, 1996; U.S. Pat. No. 5,591,616; and European PatentPublication EP 672 752 A1. Once the cells are transformed, cells havingthe inventive DNA construct incorporated in their genome may be selectedby means of a marker, such as the kanamycin resistance marker discussedabove. Transgenic cells may then be cultured in an appropriate medium toregenerate whole plants, using techniques well known in the art. In thecase of protoplasts, the cell wall is allowed to reform underappropriate osmotic conditions. In the case of seeds or embryos, anappropriate germination or callus initiation medium is employed. Forexplants, an appropriate regeneration medium is used. Regeneration ofplants is well established for many species. The resulting transformedplants may be reproduced sexually or asexually, using methods well knownin the art, to give successive generations of transgenic plants.

Polynucleotides of the present invention may also be used tospecifically suppress gene expression by methods that operatepost-transcriptionally to block the synthesis of products of targetedgenes, such as RNA interference (RNAi), and quelling. For a review oftechniques of gene suppression see Science, 288:1370-1372, 2000.Exemplary gene silencing methods are also provided in WO 99/49029 and WO99/53050. Posttranscriptional gene silencing is brought about by asequence-specific RNA degradation process which results in the rapiddegradation of transcripts of sequence-related genes. Studies haveprovided evidence that double-stranded RNA may act as a mediator ofsequence-specific gene silencing (see, e.g., review by Montgomery andFire, Trends in Genetics, 14: 255-258, 1998). Gene constructs thatproduce transcripts with self-complementary regions are particularlyefficient at gene silencing. A unique feature of thisposttranscriptional gene silencing pathway is that silencing is notlimited to the cells where it is initiated. The gene-silencing effectsmay be disseminated to other parts of an organism and even transmittedthrough the germ line to several generations.

The polynucleotides of the present invention may be employed to generategene silencing constructs and or gene-specific self-complementary RNAsequences that can be delivered by conventional art-known methods toplant tissues, such as forage grass tissues. Within genetic constructs,sense and antisense sequences can be placed in regions flanking anintron sequence in proper splicing orientation with donor and acceptorsplicing sites, such that intron sequences are removed during processingof the transcript and sense and anti-sense sequences, as well as splicejunction sequences, bind together to form double-stranded RNA.Alternatively, spacer sequences of various lengths may be employed toseparate self-complementary regions of sequence in the construct. Duringprocessing of the gene construct transcript, intron sequences arespliced-out, allowing sense and anti-sense sequences, as well as splicejunction sequences, to bind forming double-stranded RNA. Selectribonucleases bind to and cleave the double-stranded RNA, therebyinitiating the cascade of events leading to degradation of specific mRNAgene sequences, and silencing specific genes. Alternatively, rather thanusing a gene construct to express the self-complementary RNA sequences,the gene-specific double-stranded RNA segments are delivered to one ormore targeted areas to be internalized into the cell cytoplasm to exerta gene silencing effect. Gene silencing RNA sequences comprising thepolynucleotides of the present invention are useful for creatinggenetically modified plants with desired phenotypes as well as forcharacterizing genes (e.g., in high-throughput screening of sequences),and studying their functions in intact organisms.

Example 1 Isolation of cDNA Sequences from L. perenne and F. arundinaceacDNA Libraries

L. perenne and F. arundinacea cDNA expression libraries were constructedand screened as follows. Tissue was collected from L. perenne and F.arundinacea during winter and spring, and snap-frozen in liquidnitrogen. The tissues collected include those obtained from leaf blades,leaf base, pseudostem, floral stems, inflorescences, roots and stem.Total RNA was isolated from each tissue type using TRIzol Reagent (BRLLife Technologies, Gaithersburg, Md.). mRNA from each tissue type wasobtained using a Poly(A) Quik mRNA isolation kit (Stratagene, La Jolla,Calif.), according to the manufacturer's specifications. cDNA expressionlibraries were constructed from the purified mRNA by reversetranscriptase synthesis followed by insertion of the resulting cDNA inLambda ZAP using a ZAP Express cDNA Synthesis Kit (Stratagene, La Jolla,Calif.), according to the manufacturer's protocol. The resulting cDNAclones were packaged using a Gigapack II Packaging Extract (Stratagene,La Jolla, Calif.) employing 1 μl of sample DNA from the 5 μl ligationmix. Mass excision of the libraries was done using XL1-Blue MRF′ cellsand XLOLR cells (Stratagene, La Jolla, Calif.) with ExAssist helperphage (Stratagene, La Jolla, Calif.). The excized phagemids were dilutedwith NZY broth (Gibco BRL, Gaithersburg, Md.) and plated out ontoLB-kanamycin agar plates containing5-bromo-4-chloro-3-indolyl-beta-D-galactosidase (X-gal) andisopropylthio-beta-galactoside (IPTG). Of the colonies plated and pickedfor DNA preparations, the large majority contained an insert suitablefor sequencing. Positive colonies were cultured in NZY broth withkanamycin and DNA was purified following standard protocols. Agarose gelat 1% was used to screen sequencing templates for chromosomalcontamination. Dye terminator sequences were prepared using a Biomek2000 robot (Beckman Coulter Inc., Fullerton, Calif.) for liquid handlingand DNA amplification using a 9700 PCR machine (Perkin Elmer/AppliedBiosystems, Foster City, Calif.) according to the manufacturer'sprotocol.

The DNA sequences for positive clones were obtained using a PerkinElmer/Applied Biosystems Division Prism 377 sequencer. cDNA clones weresequenced from the 5′ end. The polynucleotide sequences identified asSEQ ID NO: 4, 6, 11, 127, 128 and 132 were identified from L. perenneleaf cDNA expression libraries; the polynucleotide sequences identifiedas SEQ ID NO: 1, 14, 15, 26, 32, 36, 38, 41, 49, 125, 134, 141, 144,147, and 150 were identified from L. perenne vegetative stem cDNAexpression libraries; the polynucleotide sequences identified as SEQ IDNO: 17, 22, 25, 138, and 140 were identified from L. perenne leaf andpseudostem cDNA expression libraries; the polynucleotide sequencesidentified as SEQ ID NO: 43, 57, 61, 157, and 161 were identified fromL. perenne pseudostem cDNA expression libraries; the polynucleotidesequences identified as SEQ ID NO: 10, 12, 28, 30, 34, 44, 60, 131, 133,142, 143, 145, 151, and 160 were identified from L. perenne floral stemcDNA expression libraries; the polynucleotide sequences identified asSEQ ID NO: 8, 18, 46, 52, 53, 55, 59, 136, 152, 155, 156, 159, and 162were identified from L. perenne stem cDNA expression libraries; thepolynucleotide sequences identified as SEQ ID NO: 51 and 154 wereidentified from a L. perenne root cDNA expression library; thepolynucleotide sequences identified as SEQ ID NO: 24, 27 and 139 wereidentified from L. perenne leaf blade cDNA expression libraries; thepolynucleotide sequences identified as SEQ ID NO: 9, 37, 39, 40, 45,130, 148, and 149 were identified from F. arundinacea basal leaf cDNAexpression libraries; the polynucleotide sequences identified as SEQ IDNO: 19, 21, 29, 33, 35, 47, 48, and 153 were identified from F.arundinacea combined day 3 and day 6 basal leaves cDNA expressionlibraries; the polynucleotide sequence identified as SEQ ID NO: 54 wasidentified from a F. arundinacea combined day 3 and day 6 leaves cDNAexpression library; the polynucleotide sequence identified as SEQ ID NO:56 was identified from a F. arundinacea inflorescence cDNA expressionlibrary; the polynucleotide sequences identified as SEQ ID NO: 20 and137 were identified from a subtracted F. arundinacea leaf blade cDNAexpression library; the polynucleotide sequences identified as SEQ IDNO: 7, 23, 42, 50, 62, and 129 were identified from F. arundinaceapseudostem cDNA expression libraries; the polynucleotide sequencesidentified as SEQ ID NO: 2, 13, 16 and 135 were identified from F.arundinacea leaf cDNA expression libraries; and the polynucleotidesequences identified as SEQ ID NO: 3, 5, 31, and 126 were identifiedfrom a F. arundinacea inflorescence day 0 cDNA expression library.

BLASTN Polynucleotide Analysis

The isolated cDNA sequences were compared to sequences in the EMBL DNAdatabase using the computer algorithm BLASTN. Comparisons of DNAsequences provided in SEQ ID NO: 1-62 to sequences in the EMBL DNAdatabase were made as of Oct. 19, 2001 using BLASTN algorithm Version2.0.11 [Jan. 20, 2000], and the following Unix running command: blastall-p blastn -d embldb -e 10 -G0 -E0 -r 1 -v 30 -b 30 -i queryseq -o.Comparisons of DNA sequences provided in SEQ ID NO: 125-162 to sequencesin the EMBL DNA database were made using BLASTN algorithm Version 2.2.1[Apr. 13, 2001], and the following Unix running command: blastall -pblastn -d embldb -F F -e 10 -G0 -E0 -r 1 -v 2 -b 2 -i queryseq -o.

The sequences of SEQ ID NO: 4-6, 9-11, 17-19, 21-26, 33, 44, 45, 48, 49,51-55, 59, 60, 130-132, 136, 139, 146, 151, 154-156, 159, and 162 weredetermined to have less than 50% identity to sequences in the EMBLdatabase using the computer algorithm BLASTN, as described above. Thesequences of SEQ ID NO: 2, 3, 7, 8, 14, 16, 36-38, 46, 47, 50, 56-58,61, 129, 135, 137, 138, 152, 153, 157, 158, 160 and 161 were determinedto have less than 75% identity to sequences in the EMBL database usingthe computer algorithm BLASTN, as described above. The sequences of SEQID NO: 1, 12, 13, 15, 20, 28, 31, 32, 35, 40 62, 125-128, 133, 134, 142,144 and 147 were determined to have less than 90% identity to sequencesin the EMBL database using the computer algorithm BLASTN, as describedabove. Finally, the sequences of SEQ ID NO: 29, 30, 39, 41-43, 141, 143,148, and 149 were determined to have less than 98% identity to sequencesin the EMBL database using the computer algorithm BLASTN, as describedabove.

BLASTP Polypeptide Analysis

The protein sequences corresponding to the isolated cDNA sequences werecompared to sequences in the SwissProt/Trembl protein database using thecomputer algorithm BLASTP. Comparisons of protein sequences provided inSEQ ID NO: 63-124 to sequences in the SwissProt/Trembl protein databasewere made as of Oct. 19, 2001 using BLASTP algorithm Version 2.0.11[Jan. 20, 2000], and the following Unix running command: blastall -pblastp -dstdb-e 10 -G0 -E0 -v 30 -b 30 -i queryseq -o. Comparisons ofprotein sequences provided in SEQ ID NO: 163-190 to sequences in theSwissProt/Trembl protein database were made using BLASTP algorithmVersion 2.2.1 [Apr. 13, 2001], and the following Unix running command:blastall -p blastp -d stdb -F F -e 10 -G0 -E0 -v 2 -b 2 -i queryseq -o.

The sequences of SEQ ID NO: 65-68, 72, 73, 78, 80, 81, 84, 85, 87, 88,106, 107, 110, 111, 113-115, 117, 118 and 121 were determined to haveless than 50% identity to sequences in the SwissProt/Trembl databaseusing the computer algorithm BLASTP, as described above. The sequencesof SEQ ID NO: 71, 79, 82, 83, 86, 95, 98-100, 112, 116, 120, 122-124,167, 168, 171-174, 185, 188, and 190 were determined to have less than75% identity to sequences in the SwissProt/Trembl database using thecomputer algorithm BLASTP, as described above. The sequences of SEQ IDNO: 63, 64, 69, 70, 74-77, 90, 91, 93, 94, 97, 101, 102, 104, 108, 109,119, 175, 183, 187, and 189 were determined to have less than 90%identity to sequences in the SwissProt/Trembl database using thecomputer algorithm BLASTP, as described above. Finally, the sequences ofSEQ ID NO: 89, 92, 96, 103, 105, 163-165, 169, 170, 177, 179, 181, 184,and 186 were determined to have less than 98% identity to sequences inthe SwissProt/Trembl database using the computer algorithm BLASTP, asdescribed above.

BLASTX Polynucleotide Analysis

The isolated cDNA sequences were compared to sequences in theSwissProt/Trembl protein database using the computer algorithm BLASTX.Comparisons of DNA sequences provided in SEQ ID NO: 1-62 to sequences inthe SwissProt/Trembl protein database were made as of Oct. 19, 2001using BLASTX algorithm Version 2.0.11 [Jan. 20, 2000], and the followingUnix running command: blastall -p blastx -dstdb -e 10 -G0 -E0 -v 30 -b30 -i queryseq -o. Comparisons of DNA sequences provided in SEQ ID NO:1-62 to sequences in the SwissProt/Trembl protein database were madeusing BLASTX algorithm Version 2.2.1 [Apr. 13, 2001], and the followingUnix running command: blastall -p blastx -d stdb -F F -e 10 -G0 -E0 -v 2-b 2 -i queryseq -o.

The sequences of SEQ ID NO: 11, 44, 45, 48, 49, 51, 52, 55, 130, 132,155, 156, and 162 were determined to have less than 50% identity tosequences in the SwissProt/Trembl database using the computer algorithmBLASTX, as described above. The sequences of SEQ ID NO: 3-10, 16-26, 33,36-38, 40-43, 50, 53, 54, 56, 58-62, 129, 131, 135-139, 146, 150, 151,154, and 158-161 were determined to have less than 75% identity tosequences in the SwissProt/Trembl database using the computer algorithmBLASTX, as described above. The sequences of SEQ ID NO: 1, 2, 12-15, 27,28-32, 34, 35, 39, 46, 47, 57, 125-128, 133, 134, 141-145, 147-149, 152,153, and 157 were determined to have less than 90% identity to sequencesin the SwissProt/Trembl database using the computer algorithm BLASTX, asdescribed above. Finally, the sequence of SEQ ID NO: 140 was determinedto have less than 98% identity to sequences in the SwissProt/Trembldatabase using the computer algorithm BLASTX, as described above.

The location of open reading frames (ORFs), by nucleotide position,contained within the sequences of SEQ ID NO: 1-62 and 125-162, and thecorresponding amino acid sequences are provided in Table 2 below. SEQ IDNO: 1-8, 10-15, 17, 19, 21, 23-25, 28-52, 54-59, 61-62 and 125-162 arebelieved to contain full-length ORFs.

TABLE 2 POLYNUCLEOTIDE POLYPEPTIDE SEQ ID NO: ORF SEQ ID NO: 1 56-2,02063 2 64-2,010 64 3 64-1,926 65 4 74-1,945 66 5 40-1,911 67 6 79-1,938 687 246-1,514  69 8 264-1,532  70 9 84-3,272 71 10 73-3,297 72 11129-2,942  73 12 46-2,472 74 13 113-2,539  75 14 61-2,505 76 15103-2,253  77 16  3-1,439 78 17 26-1,777 79 18  2-1,174 80 19 59-1,85281 20  2-1,201 82 21  1-1,779 83 22 198-1,097  84 23 27-1,772 85 2436-1,802 86 25 78-2,084 87 26  2-1,423 88 27  3-1,622 89 28 85-1,764 9029 72-1,751 91 30 127-1,800  92 31 137-1,810  93 32 62-1,567 94 3380-1,597 95 34 32-1,117 96 35 86-1,171 97 36 55-852   98 37 75-872   9938 149-1,240  100 39 90-1,118 101 40 28-1,110 102 41 66-1,148 103 4264-1,146 104 43 85-1,170 105 44 88-1,683 106 45 93-1,721 107 46111-2,246  108 47 144-2,285  109 48 22-993   110 49  4-1,038 111 5087-1,067 112 51 59-1,135 113 52 18-1,052 114 53 1-882  115 54 80-1,015116 55 322-1,014  117 56 172-762   118 57 118-1,299  119 58 5-595  12059 14-1,003 121 60 1-987  122 61 65-1,174 123 62 103-1,245  124 12555-2,019 163 126 63-1,925 164 127 73-1,944 165 128 71-1,930 166 13172-3,299 167 132 134-2,950  168 133 45-2,471 169 134 65-2,512 170 13574-1,819 171 136 170-1,855  172 137 28-1,770 173 138 26-1,733 174 13935-1,801 175 140 71-2,083 176 141 63-1,607 177 143 126-1,799  178 14461-1,566 179 145 67-1,152 180 147 148-1,239  181 149 27-1,109 182 15187-1,718 183 153 143-2,284  184 156 46-1,017 185 157 117-1,313  186 15881-1,193 187 159 12-1,001 188 160 26-1,018 189 162 50-1,027 190

SEQ ID NO: 25 and 163 are related to SEQ ID NO: 1 and 63, respectively;SEQ ID NO: 126 and 164 are related to SEQ ID NO: 3 and 65, respectively;SEQ ID NO: 127 and 165 are related to SEQ ID NO: 4 and 66, respectively;SEQ ID NO: 128 and 166 are related to SEQ ID NO: 6 and 68, respectively;SEQ ID NO: 129 is an extended sequence of SEQ ID NO: 7; SEQ ID NO: 130is an extended sequence of SEQ ID NO: 9; SEQ ID NO: 131 and 167 arerelated to SEQ ID NO: 10 and 72, respectively; SEQ ID NO: 132 and 168are related to SEQ ID NO: 11 and 73, respectively; SEQ ID NO: 133 and169 are related to SEQ ID NO: 12 and 74, respectively; SEQ ID NO: 134and 170 are related to SEQ ID NO: 14 and 76, respectively; SEQ ID NO:135 and 171 are full-length sequences of SEQ ID NO: 16 and 78,respectively; SEQ ID NO: 136 and 172 are full-length sequences of SEQ IDNO: 18 and 80, respectively; SEQ ID NO: 137 and 173 are related to SEQID NO: 20 and 82, respectively; SEQ ID NO: 138 and 174 are full-lengthsequences of SEQ ID NO: 22 and 84, respectively; SEQ ID NO: 139 and 175are related to SEQ ID NO: 24 and 86, respectively; SEQ ID NO: 140 and176 are related to SEQ ID NO: 25 and 87, respectively; SEQ ID NO: 141and 177 are full-length sequences of SEQ ID NO: 26 and 88, respectively;SEQ ID NO: 142 is related to SEQ ID NO: 28 and encodes the same aminoacid sequence; SEQ ID NO: 143 and 178 are related to SEQ ID NO: 30 and92, respectively; SEQ ID NO: 144 and 179 are related to SEQ ID NO: 32and 94, respectively; SEQ ID NO: 145 and 180 are full-length sequencesof SEQ ID NO: 34 and 96, respectively; SEQ ID NO: 146 is related to SEQID NO: 36 and encodes the same amino acid sequence; SEQ ID NO: 147 and181 are related to SEQ ID NO: 38 and 100, respectively; SEQ ID NO: 148is related to SEQ ID NO: 39, and encodes the same amino acid sequence;SEQ ID NO: 149 and 182 are related to SEQ ID NO: 40 and 102,respectively; SEQ ID NO: 150 is related to SEQ ID NO: 41 and encodes thesame amino acid sequence; SEQ ID NO: 151 and 183 is related to SEQ IDNO: 44 and 106, respectively; SEQ ID NO: 152 is related to SEQ ID NO:46, and encodes the same amino acid sequence; SEQ ID NO: 153 and 184 arerelated to SEQ ID NO: 47 and 109, respectively; SEQ ID NO: 154 isrelated to SEQ ID NO: 51, and encodes the same amino acid sequence; SEQID NO: 155 is related to SEQ ID NO: 52, and encodes the same amino acidsequence; SEQ ID NO: 156 and 185 are full-length sequences of SEQ ID NO:53 and 115, respectively; SEQ ID NO: 162 and 190 are variants of SEQ IDNO: 156 and 185, respectively, with a difference in the 5′ region of SEQID NO: 156 and 162; SEQ NO: 157 and 186 are related to SEQ ID NO: 57 and119, respectively; SEQ ID NO: 158 and 187 are related to SEQ ID NO: 58and 120, respectively; SEQ ID NO: 159 and 188 are full-length sequencesof SEQ ID NO: 59 and 121, respectively; SEQ ID NO: 160 and 189 arefull-length sequences of SEQ ID NO: 60 and 122, respectively; and SEQ IDNO: 161 is related to SEQ ID NO: 61 and encodes the same amino acidsequence.

Example 2 Use of Sucrose Phosphate Phosphatase to DephosphorylateSucrose-6-Phosphate

The F. arundinacea and L. perenne FaSPP and LpSPP genes (SEQ ID NO: 7and 8, respectively) share amino acid sequence identity withsucrose-6-phosphate phosphatase genes from other plant species (Lunn etal., Proc. Natl. Acad. Sci. USA 97:12914-12919, 2000). These genes wereamplified by PCR using the primers given in SEQ ID NO: 191 and 192 toadd an initiating methionine, and then cloned into the pET41a expressionplasmid (Novagen, Madison, Wis.). These primers amplified nucleotides263-1531 and 280-1548 for FaSPP and LpSPP, respectively. The resultingplasmids were transformed into E. coli BL21 cells using standardprotocols, and protein expression was induced using IPTG.

The soluble recombinant protein was assayed for its ability tospecifically dephosphorylate sucrose-6-phosphate (Suc-6-P) but notfructose-6-phosphate (Fru-6-P) using the procedure described by Lunn etal. (ibid.). The release of phosphate from Suc-6P and Fru-6-P wasmeasured using the Fiske-Subbarow method of determining inorganicphosphate (SIGMA assay kit; Sigma, St Louis, Mich.), with the change inabsorbance at 660 nm being proportional to the amount of phosphatereleased per unit time. As shown in FIG. 1, both the Festuca and LoliumSPP enzymes dephosphorylated Suc-6-P but not Fru-6-P, whereas controlpET41 extract had no activity on either substrate.

Example 3 Peroxidase Activity of Grass Enzymes Demonstrated by theirAbility to Oxidize 2,2′azino-bis.3-ethylbenzylthiazoline-6-sulfonic acid(ABTS)

A number of L. perenne or F. arundinacea genes (SEQ ID NO: 48-54) shareamino acid identity with peroxidase genes from other plant species(Hiraga et al., Plant Cell Physiol. 42:462-468, 2001). The putativeamino acid secretion signal sequence was identified by signalP analysisof the Lolium and Festuca sequences and homology to known peroxidaseproteins. Primers were designed to amplify DNA representing the matureprotein (minus signal sequence; Table 3.). These genes were amplified byPCR to add an initiating methionine and then cloned into the pET25bexpression plasmid (Novagen, Madison, Wis.). The resulting plasmid wastransformed into E. coli AD494 (DE3) pLysS cells using standardprotocols, and protein expression was induced using IPTG.

TABLE 3 SEQ ID SEQ ID NO Primers DNA bp Protein NO DNA PROT Gene SEQ IDNO: amplified codons 50 112 FaPER3 193 156–1077 24–326 194 52 114 LpPER5195 120–1052 35–344 196

The insoluble recombinant protein was solubilized and re-foldedfollowing protocols described for several recombinant Arabidopsisperoxidases (Teilum et al., Protein Exp. and Purif. 15:77-82, 1999). Theinsoluble inclusion bodies within E. coli were isolated from lysed cellsby standard protocols and the recombinant protein solubilized in 8Murea. The solubilized peroxidase protein was refolded to gain activeenzyme by diluting urea to 2M with 5 μM Heme, 0.25 mM Glutathionereduced, and 0.45 mM Glutathione oxidized, pH 8 (20 mM Tris-HCl). Therefolded protein was used directly to assay peroxidase activity.

Peroxidase activity was measured by incubating recombinant peroxidasewith pre-mixed ABTS/H₂O₂ liquid substrate (Sigma, St Louis, Mo.) andmeasuring ABTS oxidation by the increase in absorbance at 405 nm.Horseradish peroxidase of known activity (Sigma, St Louis, Mich.) wasused as a positive control and boiled samples as a negative control. Theresults provided in FIG. 2 show that FaPER3 and LpPER5 (SEQ ID NO: 50and 52, respectively) had similar activity to that of horseradishperoxidase in these assays.

Example 4 Use of Grass Fructosyltransferase Genes to Synthesize Fructans

Transformation of N. benthamiana Plants with Fructosyltransferase Genes

Sense constructs containing a polynucleotide including the coding regionof fructosyltransferase genes isolated from L. perenne Lp1-SST andLp6SFT1 (SEQ ID NO: 125 and 126, respectively) were inserted into apART27 derived binary vector and used to transform A. tumefaciensLBA4404 using published methods (see, An et al., “Binary Vectors,” inGelvin and Schilperoort, eds., Plant Molecular Biology Manual, KluwerAcademic Publishers: Dordrecht, 1988). The presence and integrity of thebinary vector in A. tumefaciens was verified by polymerase chainreaction (PCR). The primers px17 (SEQ ID NO: 207) and px18 (SEQ ID NO:208) were used to confirm the presence of the Lp1-SST construct, whereasthe primers px19 (SEQ ID NO: 209) and px 20 (SEQ ID NO: 210) were usedto confirm the presence of the Lp6-SFT-1 construct.

The A. tumefaciens containing the sense gene constructs were used totransform N. benthamiana leaf discs (Burow et al., Plant Mol Biol.Report 8:124-139, 1990). Several independent transformed plant lineswere established for the sense construct for each fructosyltransferasegene. DNA was isolated from transformed plants containing theappropriate fructosyltransferase gene construct using the QIAGEN DNAeasyPlant Mini Kit (Qiagen, Valencia, Calif.). Presence of thefructosyltransferase gene was verified using PCR experiments as shown inFIGS. 3 and 4. For the Lp6-SFT1 gene, the forward and reverse primersgiven in SEQ ID NO: 197 and 198 were used, respectively. These primersamplify nucleotides 1572-1980 of the Lp6-SFT1 gene which corresponds toa 406 base pair fragment. For Lp1-SST, the forward and reverse primersgiven in SEQ ID NO: 199 and 200 were used, respectively. These primersamplify nucleotides 1332-1740 of Lp1-SST, corresponding to a 414 basepair fragment.

Effects of Fructosyltransferase Genes on FructosyltransferaseConcentration in Transformed Plants

Fructans are not normally found in N. benthamiana plants; hence, ifintroduction of the sense fructosyltransferase constructs wassuccessful, it should be possible to extract fructans from thetransformed plants. The concentration of fructosyltransferase in thetransformed plants was determined using the Fructan Assay Kit (MegazymeInternational Ireland Ltd, Wicklow, Ireland). Briefly, 300 mg of leafmaterial from the independent transformed plant lines containing thefructosyltransferase sense constructs were extracted individually at 80°C. with 1 ml 80% ethanol, followed by two 1 ml extractions with water.The ethanol and water extracts were combined and frozen overnight at−20° C. Extracts were centrifuged at 20,000 g to pellet chlorophyll.Clarified extracts were treated with 1% PVP-40 to precipitate phenoliccompounds. These extracts were then reduced in volume by rotaryevaporation.

Fructan levels were determined in these extracts using the MegazymeFructan Assay kit. Briefly, sucrose, starch and reducing sugars areremoved from the plant carbohydrate extracts by using sucrase,β-amylase, pullulanase and maltase, and then converting the resultingreducing sugars to sugar alcohols. The remaining fructans are hydrolyzedwith fructanase and the reducing sugars produced (glucose and fructose)are measured by the 4-hydroxybenzoic acid hydrazide (PAHBAH) reducingsugar method. The final extracts are assayed for absorbance at 410 nm.As shown in FIG. 5, fructans could be detected in both the Lp1-SST andLp6-SFT-1 transgenic lines. Fructan levels were highest in lines 07, 09and 12 for Lp1-SST, and lines 05 and 12 for Lp6SFT-1.

Example 5 Use of Sucrose Phosphate Synthase Enzymes to SynthesizeSucrose

A F. arundinacea gene (FaSPS-N; SEQ ID NO: 9) has been identified thatshares amino acid sequence identity with sucrose phosphate synthase(SPS) from other plant species. SEQ ID NO: 7 and 8 are also SPSsequences, with SEQ ID NO: 7 being a Lolium perenne homologue of SEQ IDNO: 9. The FaSPS-N was cloned into the pcDNA3 mammalian expressionplasmid and the resulting plasmid transfected into 293T mammalian cells(human embryonic kidney derived cells) using Lipofectamine 2000 reagent(Invitrogen, Carlsbad, Calif.).

Cell lysates from transfected cells were deionized on G25 spin columnsand used in a sucrose synthesis assay. In this assay, mammalian cellextracts were tested for their ability to synthesize sucrose fromfructose-6-phosphate and uridine 5′-diphosphoglucose. Following thesynthesis reaction, hexoses were converted to sugar alcohols by boilingin the presence of 30% KOH. The sucrose synthesized was detected by theaddition of 1.4% anthrone reagent in H₂SO₄ and incubating at 40° C. for20 min. The change in absorbance at 620 nm is relative to sucrose in thereaction (Botha and Black, Aust. J. Plant Physiol. 27:81-85, 2000). Inthese experiments, introducing FaSPS-N alone into mammalian cellsproduced a sucrose synthesis activity that was not detected innon-transfected cells (FIG. 6).

A known cofactor for SPS is SPP. To test whether SPP is required for SPSactivity, the L. perenne LpSPP gene (SEQ ID NO: 8) was cloned into thepcDNA3 mammalian expression plasmid. Both the FaSPS-N and LpSPP plasmidswere co-transfected into 293T mammalian cells using Lipofectamine 2000reagent (Invitrogen, Carlsbad, Calif.). Cell lysates from transfectedcells were deionized on G25 spin columns and used in a sucrose synthesisassay as described above. As shown in FIG. 6, adding SPP did notsignificantly enhance or alter the sucrose synthesis activity of thecell extracts.

Example 6 Use of Soluble Sucrose Synthase Enzymes to Cleave Sucrose

A F. arundinacea gene (FaSUS-1; SEQ ID NO: 13) was identified thatshared amino acid sequence identity with soluble sucrose synthaseenzymes (SUS) from other plant species. Three other soluble sucrosesynthases were also identified (SEQ ID NO: 12, 14 and 15) with SEQ IDNO. 12 being a direct homologue from L. perenne. The FaSUS-1 gene wascloned into the pcDNA3 mammalian expression plasmid, which wastransiently transfected into 293T mammalian cells (human embryonickidney derived cells) using Lipofectamine 2000 reagent (InvitrogenCarlsbad, Calif.). Transfected cells were grown for several days beforeharvesting (by scraping cells in a sucrose synthase buffer). Harvestedcells were frozen on dry ice and freeze-thawed twice before pelletingcell debris by centrifugation. The resulting supernatant (cell lysate)was deionized on G25 spin columns and then used in a sucrose cleavageassay as described by Sebkova et al. (Plant Physiol. 108:75-83, 1995).In these assays, the cell lysates were tested for their ability tocleave sucrose in the presence of UDP to produce fructose and uridine5′-diphosphoglucose. Following a 30 min incubation at 30° C., the enzymeactivity was stopped by boiling the tubes for 1 min. Both NAD andUDP-glucose dehydrogenase were added and the change in OD at 340 nM(production of NADPH) was measured. As shown in FIG. 7, significantlyhigher levels of sucrose cleavage were observed in cells transfectedwith FaSUS1 construct than in non-transfected control cells.

Example 7 Use of Acid Invertases to Cleave Sucrose

A number of acid (vacuolar and cell wall) invertase genes from L.perenne and F. arundinacea (SEQ ID NO: 17, 19, 21, 23 and 135-141) wereidentified that share amino acid sequence identity with acid invertasesfrom other plant species (Unger et al., Plant Physiol. 104:1351-1357,1994; Goetz and Roitsch, J. Plant Physiol. 157:581-585, 2000). Thesesequences were analysed by SignalP and homology to identify signalregions and propeptide sequences, and primers were designed to amplifythe DNA sequence encoding the mature protein (Table 4).

TABLE 4 SEQ ID SEQ NO ID NO Primers SEQ DNA bp Protein DNA PROT Gene IDNO amplified codons 17 79 LpCWINV1 201 137–1803 38–583 202 19 81FaCWINV4 203 134–1912 26–597 204 25 87 LpSINV1 205 387–2124 104–668  206

The PCR fragments were cloned into pPICZαA vectors for expression inmethylotrophic yeast Pichia pastoris (EasySelect™ Pichia Expression Kit,Invitrogen, Carlsbad, Calif.). The sequences were cloned in frame withthe α-mating factor for secretion of the recombinant invertase proteininto liquid media, following similar methods described for theexpression of barley 6-SFT and fescue 1-SST in P. pastoris (Hochstrasseret al., FEBS Letters 440:356-360, 1998; Lüscher et al., Plant Physiol.,124:1217-1227, 2000). The media was concentrated 10 fold by Vivaspin 30kDa spin column (VivaScience, Hannover, Germany) to concentraterecombinant protein and used directly to assay invertase activity.Recombinant protein was assayed with 100 mM sucrose in 500 μl phosphatebuffer pH5.0, at 30° C. for 1 hour. Release of glucose by invertaseactivity was measured using a glucose HK assay kit (Sigma, St Louis,Mo.). FIG. 8 shows the glucose released by invertase activity in termsof glucose concentration in the assay mix. As shown in FIG. 8, invertaseactivity was observed for the vacuolar invertase (LpSINV1; SEQ NO: 25)and the two cell wall invertases (LpCWINV1 and FaCWINV4; SEQ NO: 17 and19, respectively) but not for an empty vector (pPICZalphaA) control.

Example 8 Use of Tannin Genes to Modify Tannin Biosynthesis

Certain Arabidopsis mutants of the transparent testa (tt) phenotype donot make the anthocyanin pigment cyanidin and therefore have no seedcoat color. The genes responsible for many of these mutants have nowbeen identified as shown in Table 5.

TABLE 5 Enzyme Abbreviation Locus Chromosome Dihydroflavanol-4-reductaseDFR tt3 5 Chalcone synthase CHS tt4 5 Chalcone isomerase CHI tt5 3Flavanone 3-β-hydroxylase F3βH tt6 3

Over-expression of the maize genes for CHS, CHI and DFR has been shownto complement the Arabidopsis tt4, tt5 and tt3 mutants, respectively,thereby restoring cyanidin synthesis and seed coat color (Dong et al.,Plant Physiol. 127:46-57, 2001). Complementation of these Arabidopsismutants may therefore be employed to demonstrate the function of theinventive polynucleotides encoding enzymes involved in the tanninbiosynthetic pathway.

Sense constructs containing a polynucleotide including the coding regionof tannin genes isolated from L. perenne or F. arundinacea LpCHS, LpCHI,LpF3βH, LpDFR1, FaCHI and FaF3βH (SEQ ID NO: 157, 55, 161, 159, 56 and62, respectively) under the control of the CaMV 35S promoter wereinserted into a binary vector and used to transform Agrobacteriumtumefaciens LBA4404 using published methods (see, An G, Ebert P R, MitraA, Ha S B, “Binary Vectors,” in Gelvin S B, Schilperoort R A, eds.,Plant Molecular Biology Manual, Kluwer Academic Publishers: Dordrecht,1988). The presence and integrity of the binary vector in A. tumefacienswas verified by polymerase chain reaction (PCR) using the primer pairsdescribed in Table 6.

TABLE 6 Transparent Forward Primer Reverse Primer Gene SEQ ID NO: testaline SEQ ID NO: SEQ ID NO: LpCHS 157 tt4 211 212 LpCHI 55 tt5 213 214LpF3βH 161 tt6 217 218 LpDFR1 159 tt3 215 216 FaCHI 56 tt5 213 214FaF3βH 62 tt6 217 218

The A. tumefaciens containing the sense gene constructs are used totransform Arabidopsis by floral dipping (Clough and Bent, Plant J.16:735-743, 1998). Several independent transformed plant lines wereestablished for the sense construct for each of the tannin genes.Specifically, LpDFR1 constructs were transformed into Arabidopsis tt3mutants, LpCHS constructs were transformed into Arabidopsis tt4 mutants,LpCHI and FaCHI constructs were transformed into Arabidopsis tt5mutants, and LpF3βH and FaF3βH constructs were transformed intoArabidopsis tt6 mutants. Several independent transformed plant lineswere established for the construct for each of the tannin genes.Transformed plants containing the appropriate tannin gene construct wereverified using PCR.

The presence of cyanidin in the FaCHI transformed plants is demonstratedby a phenotypic change in plant seedling color and by analyzing cyanidinextracts made from transgenic plants grown under stressed conditions(Dong et al., Plant Physiol. 127:46-57, 2001). Briefly, cyanidins areextracted from plant tissue with an acid/alcohol solution (HCl/methanol)and water. Chlorophyll is removed by freezing the extracts followed bycentrifugation at 4° C. at 20,000 g for 20 min. Any remainingchlorophyll is removed through a chloroform extraction. The absorbanceat 530 nm is measured for each of the cyanidin extracts. Non-transgenicwild type and control Arabidopsis plants are used as controls.

SEQ ID NO: 1-218 are set out in the attached Sequence Listing. The codesfor nucleotide sequences used in the attached Sequence Listing,including the symbol “n,” conform to WIPO Standard ST.25 (1998),Appendix 2, Table 1.

All references cited herein, including patent references and non-patentpublications, are hereby incorporated by reference in their entireties.

While in the foregoing specification this invention has been describedin relation to certain preferred embodiments, and many details have beenset forth for purposes of illustration, it will be apparent to thoseskilled in the art that the invention is susceptible to additionalembodiments and that certain of the details described herein may bevaried considerably without departing from the basic principles of theinvention.

1. A method of producing a plant cell or plant with altered lignincomposition, the method comprising down-regulating expression of anendogenous polypeptide with at least 98% identity to the sequence of SEQID NO: 107 or SEQ ID NO: 183, wherein the polypeptide has ferulate5-hydroxylase (F5H) activity, thereby altering lignin composition. 2.The method of claim 1, wherein the polypeptide has at least 98% identityto the sequence of SEQ ID NO:
 107. 3. The method of claim 1, wherein thepolypeptide has the sequence of SEQ ID NO:
 107. 4. The method of claim1, wherein the polypeptide has at least 98% identity to the sequence ofSEQ ID NO:
 183. 5. The method of claim 1, wherein the polypeptide hasthe sequence of SEQ ID NO:
 183. 6. The method of claim 1, whereindown-regulation is effected by introducing into the plant cell or plant,a polynucleotide sequence capable of hybridizing under stringenthybridization conditions to: (a) an endogenous polynucleotide thatencodes the polypeptide, or (b) the complement of the endogenouspolynucleotide that encodes the polypeptide, wherein the stringenthybridization conditions comprise prewashing in a solution of 6×SSC,0.2% SDS; hybridizing at 65° C., 6×SSC, 0.2% SDS overnight; followed bytwo washes of 30 minutes each in 1×SSC, 0.1% SDS at 65° C. and twowashes of 30 minutes each in 0.2×SSC, 0.1% SDS at 65° C.
 7. The methodof claim 6, wherein the polynucleotide sequence is capable ofhybridizing to a coding region of the endogenous polynucleotide.
 8. Themethod of claim 6, wherein the polynucleotide sequence is capable ofhybridizing to a non-coding region of the endogenous polynucleotide. 9.The method of claim 6, wherein the polynucleotide sequence iscomplementary to part of the endogenous polynucleotide.
 10. The methodof claim 6, wherein the polynucleotide sequence comprises a sequencewith 98% identity to SEQ ID NO:
 45. 11. The method of claim 6, whereinthe polynucleotide sequence comprises the sequence of SEQ ID NO:
 45. 12.The method of claim 6, wherein the polynucleotide sequence comprises asequence with 98% identity to SEQ ID NO:
 151. 13. The method of claim 6,wherein the polynucleotide sequence comprises the sequence of SEQ ID NO:151.
 14. The method of claim 6, wherein the polynucleotide sequence isstably incorporated into the genome of the plant.
 15. The method ofclaim 6, wherein the polynucleotide sequence is expressed inside theplant.
 16. The method of claim 6, wherein the polynucleotide sequence isintroduced into the plant as pail of a genetic construct.
 17. The methodof claim 6, wherein the nucleic acid is introduced into the plant aspart of a genetic construct comprising: (a) a promoter; and (b) thepolynucleotide sequence.
 18. The method of claim 17, wherein the nucleicacid is in an antisense orientation relative to the promoter.
 19. Themethod of claim 1, wherein the plant is a grass.
 20. The method of claim19, wherein the plant is selected from the group consisting of Loliumperenne and Festuca arundinacea.